Therapy with clostridium perfringens enterotoxin to treat ovarian and uterine cancer

ABSTRACT

The invention discloses high levels of receptors for  Clostridium perfringens  enterotoxin (CPE) have been found in ovarian cancer and uterine cancer tissue samples. In addition, successful in vivo treatment of a mouse model of ovarian cancer with intraperitoneal injection of CPE is disclosed. High levels of Ep-CAM protein is also disclosed in ovarian cancer tissue samples. Thus, the invention provides a method of treating ovarian cancer and uterine cancer by administering CPE. The invention also provides a method of treating cancer in a mammal involving intraperitoneal administration of CPE, where at least some cancerous cells are located in or adjacent to the peritoneal cavity of the mammal. The invention also provides a method of treating ovarian cancer involving administering an anti-Ep-CAM antibody. The invention also provides a method of treating cancers expressing claudin-3 or claudin-4 by administering an antibody against claudin-3 and/or an antibody against claudin-4. The invention also provides a method of protecting a mammal from CPE toxicity involving administering a protective agent that binds to claudin-3 and/or claudin-4 and inhibits CPE binding to claudin-3 and/or claudin-4.

This application claims priority under 35 U.S.C. § 120 to U.S.provisional patent application Ser. No. 60,618,653, “Therapy withClostridium Perfringens Enterotoxin to Treat Ovarian and UterineCancer,” filed Oct. 14, 2004.

BACKGROUND OF THE INVENTION

Ovarian carcinoma remains the cancer with the highest mortality rateamong the gynecological malignancies, with 25,400 new cancer casesestimated in 2003 in the United States alone. Ovarian serous papillarycarcinoma (OSPC) is the most common histologic type of ovariancarcinoma. Because of the insidious onset of the disease and the lack ofreliable screening tests, two-thirds of patients have advanced diseasewhen diagnosed; and although many patients with disseminated tumorsrespond initially to standard combinations of surgical and cytologicaltherapy, nearly 90% will develop recurrence and succumb to theirdisease.

Uterine cancer is the most prevalent gynecological tumor in women, withan estimated 40,100 cases and 6,800 deaths in the United States in 2003.On the basis of clinical and histopathological variables, two subtypesof endometrial carcinoma, Type I and II tumors, have been described(1,2). Type I endometrial cancers, which account for about 80% of cases,are usually well differentiated and endometrioid in histology. They arediagnosed predominantly in younger women, and have a favorableprognosis. Type II endometrial cancers are poorly differentiated tumors,often with serous papillary or clear cell histology. Although type IItumors account for only a minority of endometrial carcinoma cases, about50% of all relapses occur in this group.

Uterine serous papillary carcinoma (USPC) tumors represent the mostaggressive variant of Type II endometrial cancer and may constitute upto 10% of endometrial tumors (3-10). The microscopic criteria fordiagnosis of USPC were first outlined by Hendrickson in 1982 (10).Typically, the neoplastic epithelium is characterized by serousdifferentiation with psammoma bodies present and with predominantlypapillary architecture (11). Pleomorphism, grade III nuclear atypia withprominent nucleoli and vesicular chromatin pattern, as well as a highmitotic activity are commonly detected in this tumor. Clinically, USPChas a propensity for intraabdominal and lymphatic spread even atpresentation and is characterized by a highly aggressive biologicbehavior (3-10, 12). USPC is a chemoresistant disease from onset, withresponses to combined cisplatinum-based chemotherapy of about 20% andshort duration (7-9). The survival rate is dismal, even when USPC isonly a minor component of the histologically more common endometrioidadenocarcinoma, and widespread metastasis and high mortality may occureven in those cases in which tumor is confined to the endometrium or toan endometrial polyp (4, 6, 12). The overall 5-year survival is about30% for all stages and the recurrence rate after surgery is extremelyhigh (50% to 80%).

Pancreatic cancer is the fifth leading cause of cancer death in the U.S.and has one of the highest mortality rates of any malignancy.

Chemotherapy resistance is a major problem in treatment of ovarian,uterine, and pancreatic cancers, and other cancers. Often a patientinitially responds to chemotherapy, but the tumor develops resistanceand recurs. New treatments for chemotherapy resistant tumors are needed.

Claudin-3 and claudin-4 are proteins found on the surface of cells ofvarious tissue types involved in tight junctions connecting one cell tothe adjacent cells (13). Claudins 3 and 4 have been found to be thereceptors for Clostridium perfringens enterotoxin, a bacterial toxinthat causes food poisoning (14-16).

Claudin-3 mRNA is expressed in normal prostrate, colon, small bowel, andpancreas (17). Claudin-4 is expressed at high levels in colon, and atmoderate levels in prostate, placenta, lung, pancreas, and lower levelsin small bowel, kidney, and uterus (17). Claudin-3 mRNA expression inprostate adenocarcinoma was found to be equal to or greater thanexpression in surrounding normal prostate tissue (17). The prostatecancer cells in tissue culture were sensitive to killing by Clostridiumperfringens enterotoxin (CPE) (17).

In another report, claudin-4 mRNA was found to be not expressed orweakly expressed in normal pancreas, but was highly expressed in mostprimary pancreatic carcinoma samples (18). Claudin-4 expression was alsofound in colon cancer, breast cancer, and gastric cancer samples (18).CPE was reported to kill pancreatic cancer cell lines in vitro (18).Pancreatic tumor xenografts were induced in nude mice, and the tumorswere directly injected with CPE over the course of 5 days. TheCPE-treated tumors showed no increase in size and exhibited areas ofnecrosis, while the untreated tumors grew (18).

Hough et al. reported results of serial analysis of gene expression(SAGE) comparing expression of genes in ovarian surface epithelium,cystadenoma, and ovarian primary tumors (19). Many genes were found tobe upregulated in ovarian tumors compared to normal ovarian epithelium.Several of these were surface proteins. Surface proteins shown to beupregulated included claudin-3 and -4, HE4, mucin-1, epithelial cellularadhesion molecule, and mesothelin (19). Claudin-3 and -4 mRNA andprotein levels were also reported to be elevated in ovarian carcinomasamples as compared to normal ovarian tissue and ovarian cystadenoma(20).

New methods for treating cancer are needed. Methods for treating themost deadly cancers, including ovarian cancer, pancreatic cancer, anduterine serous papillary carcinoma, are particularly needed. Methods fortreating chemotherapy resistant cancers are particularly needed.Preferably the methods would have reduced side effects and involvereduced toxicity for healthy non-target tissue.

SUMMARY

The invention is based in part on the discovery that expression ofclaudin-3 and -4 is higher in metastatic ovarian cancer cells than cellsfrom primary ovarian tumors, and higher in chemotherapy-resistantovarian cancer cells than in chemotherapy-naive ovarian cancer cells.Thus Clostridium perfringens enterotoxin (CPE) is well suited for use intreating ovarian cancer patients whose tumors have spread or resistedchemotherapy.

It has also been discovered that uterine serous papillary carcinoma(USPC) cells express 12-times more claudin-4 and 8-times more claudin-3than healthy uterine epithelial cells.

It is demonstrated herein that ovarian cancer can be treatedsuccessfully in vivo in a mouse model with administration of CPE. Thus,one aspect of the invention is treating uterine or ovarian cancer,particularly metastatic or chemotherapy resistant forms of uterine orovarian cancer, in vivo in a mammal by administering CPE.

One aspect of the invention is administering CPE intraperitoneally totreat tumors found in or adjacent to the peritoneal cavity. This reducesthe toxicity of CPE administration because it spares tissues distantfrom the peritoneal cavity. That allows higher doses to be administered,increasing the efficacy against tumors in or near the peritoneal cavity.

Another aspect of the invention involves administering to non-targettissues an agent that protects cells against CPE toxicity in conjunctionwith intraperitoneal administration of CPE. This further protects thenon-target tissues, reducing the side effects of CPE and allowing theuse of higher doses of CPE. One such protective agent is a carboxyterminal fragment of the CPE protein.

Thus, one embodiment of the invention provides a method of treatingcancer in a mammal involving: administering to the mammal atherapeutically effective amount of CPE or a pharmaceutically effectivesalt thereof, wherein the cancer is ovarian cancer or uterine cancer.

Another embodiment of the invention provides a method of treating cancerin a mammal involving: intraperitoneally administering to the mammal atherapeutically effective amount of CPE or a pharmaceutically acceptablesalt thereof. This method is suitable when at least some cancerous cellsare located in or adjacent to the peritoneal cavity of the mammal, andthe cells are sensitive to CPE.

One embodiment of the invention provides a method of determining thesensitivity of a gynecological malignancy to CPE involving detecting thepresence or absence of claudin-3 and/or claudin-4 in a tissue samplecomprising a portion of the malignancy.

One embodiment of the invention provides a use of CPE to prepare amedicament effective to treat ovarian cancer or uterine cancer in amammal.

One embodiment of the invention provides a use of CPE to prepare amedicament effective to treat cancer in a mammal, wherein the medicamentis adapted for intraperitoneal administration.

It is also reported herein that the TROP-1/Ep-CAM gene and the Ep-CAMprotein are overexpressed in ovarian serous papillary carcinoma (OSPC)compared to normal ovarian epithelium (NOVA). The TROP-1/Ep-CAM mRNA isexpressed 39-fold higher in OSPC than in NOVA. Thus, antibodies againstEp-CAM can be an effective treatment for ovarian cancer.

Accordingly, one embodiment of the invention provides a method oftreating cancer in a mammal involving administering to the mammal atherapeutically effective amount of an anti-Ep-CAM antibody.

Another embodiment of the invention is a method of determining thesensitivity of a gynecological malignancy to treatment with ananti-Ep-CAM antibody involving detecting the presence or absence ofEp-CAM in a tissue sample comprising a portion of the malignancy.

Another embodiment of the invention provides a method of protecting amammal from CPE toxicity involving administering a non-toxic agent thatbinds to claudin-3 and/or claudin-4 and inhibits CPE binding toclaudin-3 and/or claudin-4.

Another embodiment of the invention provides a method of treating cancerin a mammal involving administering to the mammal an antibody againstclaudin-3 and/or claudin-4, wherein cancerous cells in the mammalcontain claudin-3 and/or claudin-4.

Another embodiment of the invention provides a humanized antibodyagainst an extracellular portion of claudin-3 and/or an extracellularportion of claudin-4.

Another embodiment of the invention provides an antibody that includesan antigen-binding region comprising residues 290-319 of SEQ ID NO:1 ora fragment thereof that binds specifically to claudin-3 and/orclaudin-4. The fragment may include residues 315-319 of SEQ ID NO:1

Another embodiment of the invention provides a method of treating cancerin a mammal involving: administering to the mammal a therapeuticallyeffective amount of a cytotoxic agent that binds specifically toclaudin-3 and/or claudin-4; wherein the cancer is ovarian cancer oruterine cancer.

Another embodiment of the invention provides an anticancer agentcontaining: (a) a moiety that binds specifically to claudin-3 and/orclaudin-4; coupled to (b) a cytotoxic moiety; wherein the agent iseffective to kill tumor cells overexpressing claudin-3 and/or claudin-4in vitro or in vivo, and wherein the agent is not CPE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing hierarchical clustering of 15 primaryovarian cell lines (10 OSPC and 5 NOVA) and 2 established OSPC celllines (UCI-101 and UCI-107).

FIG. 2 shows bar graphs of quantitative real-time PCR and microarrayexpression analyses of TROP-1, CD24, claudin-3, and claudin-4 genesdifferentially expressed between OSPC and NOVA.

FIG. 3 shows representative FACS analyses of CD24 staining (left panel)and TROP-1/Ep-CAM staining (right panel) of 2 primary OSPC cell linesand 1 NOVA cell line. Data on CD24 and TROP-1/Ep-CAM are shown in solidblack, while isotype control MAb profiles are shown in white.

FIG. 4 depicts an unsupervised hierarchical clustering of fifteenprimary uterine cell lines (10 USPC and 5 NEC).

FIG. 5 shows bar graphs of quantitative RT-PCR and microarray expressionanalysis of CDKN2A/p16, CDKN2A/p14ARF, L1CAM, claudin-3, claudin-4,GRB-7, and c-erbB2 genes differentially expressed between USPC and NEC.

FIG. 6. Quantitative RT-PCR analysis of claudin-3 and claudin-4expression. The Y axis represents the fold induction relative to normalovary expression (sample 1). The X axis represents each sample testedfor claudin-3 (A) and claudin-4 (B). The first 15 bars represent normalovarian epithelium (1-3), normal endometrial epithelium (4-6), normalcervical keratinocytes (7), primary squamous cervical cancer cell lines(8-10), primary adenocarcinoma cervical cancer cell lines (11-13),Epstein-Barr transformed B lymphocytes (LCL) (14), and human fibroblasts(15). The following 16 bars represent primary ovarian cancer cell lines(16-21 serous papillary ovarian cancers, 22-26 clear cell ovariantumors) and established serous ovarian cancer cell lines, 27-31 (i.e,UCI 101, UCI 107, CaOV3, OVACAR-3, and OVARK-5).

FIG. 7. Quantitative RT-PCR analysis of claudin-3 and claudin-4expression in chemotherapy naïve versus chemotherapy resistant/recurrentovarian cancer. The Y axis represents the fold induction relative tonormal ovary expression. The X axis represents each sample tested forclaudin-3 and claudin-4. FIGS. 7A and 7B: 1 (chemotherapy naïve ovariancancers=6 OSPC samples, mean±SEM); 2 (chemotherapy resistant/recurrentovarian cancer=6 OSPC samples, mean±SEM) (p<0.05). FIGS. 7C and 7D: 1(chemotherapy naïve) and 2 (chemotherapy resistant) represent claudin-3and claudin-4 expression in autologous matched OVA-1 tumors. 3(chemotherapy naïve) and 4 (chemotherapy resistant) represent claudin-3and claudin-4 expression in autologous matched OVA-4 tumors. 5(chemotherapy naïve) and 6 (chemotherapy resistant) represent claudin-3and claudin-4 expression in autologous matched OVA-6 tumors.

FIG. 8. Quantitative RT-PCR analysis of claudin-3 and claudin-4expression in primary vs. metastatic ovarian cancer tumors. The Y axisrepresents the fold induction of claudin-3 (left panel) and claudin-4(right panel) relative to normal ovary expression. The sample tested isindicated on the X axis as (1) OVA-1 primary tumor, and (2) OVA-1metastatic tumor.

FIG. 9. Representative dose dependent CPE-mediated cytotoxicity ofprimary ovarian cancers compared to positive control Vero cells ornegative controls (i.e., normal and neoplastic cells) after 24 hrsexposure to CPE. VERO: positive control cells. OVA-1 to OVA-6: primaryovarian tumors. OVARK-5, CaOV3, OVACAR-3: established serous ovariantumors. Nova: normal ovarian epithelium. Norm CX: normal cervixkeratinocytes. Fibroblast: normal human fibroblasts. LCL: lymphoblastoidB-cells. PBL: normal peripheral blood lymphocytes. CX1-3: primarysquamous cervical cancer. ADX1-3: primary adenocarcinoma cervicalcancer.

FIGS. 10A and B. Survival of C.B-17/SCID Mice after i.p. injection of5-7.5×10⁶ viable OVA-1 tumor cells. Animals harboring four-week (A) andone-week (B) established OVA-1 tumors were injected i.p. with doses ofCPE ranging from 5 to 8.5 μg as described in the material and methods.CPE was administered i.p. every 72 hrs until death or end of study. Micewere evaluated on a daily basis and sacrificed when moribund. In bothstudies, the log-rank test yielded p<0.0001 for the differences insurvival.

DETAILED DESCRIPTION Definitions

The term “Clostridium perfringens enterotoxin” (CPE) includes the nativeenterotoxin (SEQ ID NO:1) or an engineered, mutant, or variantenterotoxin that is toxic and binds specifically in vivo in a mammal toclaudin-3 and/or claudin-4. The portion of native CPE responsible fortoxicity is thought to be approximately residues 45-116 of full-lengthnative CPE (SEQ ID NO:1). The portion of native CPE responsible forbinding to claudin-3 and claudin-4 is residues 290-319 of SEQ ID NO:1 ora portion thereof (23-27).

The term “chemotherapy-resistant” cancer cells refers to cells of atumor that has recurred after chemotherapy, not responded tochemotherapy, has demonstrated survival in vitro or in vivo afterexposure to doses of one or more chemotherapy agents that wouldotherwise be considered for use against the tumor, or is of a type oftumor (such as USPC) that, based on data from experimental animals orother patients, is considered to not respond to chemotherapy or recurafter chemotherapy.

The term “metastasized” or “metastatic” refers to cancer cells that havespread to a different part of the body from the site where the primarytumor for the cancer originated.

The term “therapeutic index” is defined herein as the ratio of theminimal toxic dose (LD₅₀) to the minimal dose that induces a measurableresponse in vivo, such as arresting tumor growth.

The term “cytotoxic agent” refers to an agent that kills cancerous cellsin vitro or in vivo in a mammal. The term “cytotoxic agent” includesagents that are not toxic in the absence of immune system components butthat promote an immune response that kills cancerous cells in vivo.

The term “Fc” refers to the constant portion of an immunoglobulinprotein.

The terms “pancreatic cancer,” “ovarian cancer,” “uterine cancer,” etc.refer to the organ or tissue type in which the primary tumor of thecancer originated.

DESCRIPTION

CPE is an enterotoxin responsible for many cases of food poisoning.Native CPE is a 319 residue protein (SEQ ID NO:1) that bindsspecifically to claudin-3 and claudin-4 on the surface of certain cellsin the gastrointestinal tract. After CPE binds to its target cellreceptor (claudin-3 or claudin-4) a 90 kDa small membrane complex isformed in the target cell plasma membrane containing intact CPE.Additional target cell proteins then associate with the small complex toform a large 160 kDa membrane complex that allows partial insertion ofCPE into the membrane, allowing small molecule permeability changes andlysis (21, 22).

Functional domain mapping of the full-length 319 amino acid CPE proteinshowed that a CPE (290-319) COOH-terminus fragment is sufficient forhigh affinity binding to receptors and small complex formation, but doesnot initiate large complex formation and cytolysis. In fact, CPE(290-319) fragment inhibits cytolysis by full-length CPE (26). Deletionfrom full-length native CPE of the five residues at the C-terminus,residues 315-319, is enough to abolish binding to the receptors (25).Approximately residues 45-116 of CPE are essential for large complexformation and cytolysis. With NH2-terminal residues 1-44 deleted, CPE(45-319) is actually more toxic than full-length CPE (23-27).

Thus, in particular embodiments of the invention, the CPE is a variantCPE comprising residues 45-319 of SEQ ID NO:1. In other particularembodiments, the CPE comprises a sequence at least 80% identical, or atleast 90% identical to residues 45-319 of SEQ ID NO:1.

In particular embodiments of the invention, the CPE comprises residues290-319 of SEQ ID NO:1. In particular embodiments, the CPE comprises afragment of residues 290-319 of SEQ ID NO:1 that binds specifically toclaudin-3 and/or claudin-4. In particular embodiments, the CPE comprisesresidues 315-319 of SEQ ID NO:1. In particular embodiments, CPEcomprises a variant of residues 290-319 of SEQ ID NO:1 that specificallybinds claudin-3 and claudin-4. In particular embodiments, CPE comprisesa variant of residues 290-319 of SEQ ID NO:1 that specifically bindsclaudin-3 or claudin-4.

In particular embodiments, the CPE binds claudin-3 and/or claudin-4 witha K_(D) of less than 10 μM, or more preferably less than about 1 μM,most preferably less than about 0.1 μM. This can be assayed as describedin references 14 and 26.

One embodiment of the invention is a method of treating ovarian canceror uterine cancer in a mammal involving administering a therapeuticallyeffective amount of CPE or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the cancer includes chemotherapy-resistantmalignant cells. As shown below, chemotherapy-resistant ovarian cancercells have higher expression of claudin-3 and -4 than chemotherapy-naiveovarian cancer cells. USPC, a chemotherapy-resistant and aggressive formof uterine cancer also is shown below to have high expression ofclaudin-3 and -4. Thus, CPE is particularly suited for treatment ofovarian and uterine cancer. Moreover, since CPE is a toxin, oncologistsmay be hesitant to use it on a tumor that is thought to be sensitive toless toxic conventional chemotherapy agents.

In another particular embodiment of the method, the cancer includesmetastatic malignant cells. It is shown below in Example 3 thatmetastatic ovarian tumors express higher levels of claudin-3 and -4 thanprimary ovarian tumors. USPC, an aggressive form of uterine cancer thattends to readily metastasize also is shown below to have high expressionof claudin-3 and -4.

In a particular embodiment, the cancer is uterine serous papillarycarcinoma (USPC).

In a particular embodiment, the cancer is ovarian serous papillarycarcinoma (OSPC).

In a particular embodiment of the method, the CPE or pharmaceuticallyacceptable salt thereof is administered intraperitoneally.

The dosage of CPE or a pharmaceutically acceptable salt thereof forintraperitoneal administration is 0.2 to 0.8 mg/kg body mass, in aparticular embodiment.

In other particular embodiments, the CPE or pharmaceutically acceptablesalt thereof is administered intravenously or intratumorally.

In particular embodiments, the CPE or pharmaceutically acceptable saltthereof has a therapeutic index as administered of at least 5. Thetherapeutic index can depend on the route of administration. Thus, fortumors located in or adjacent to the peritoneal cavity, intraperitonealadministration is likely to have a higher therapeutic index thansystemic intravenous administration. The therapeutic index will alsodepend on the type of cancer. The therapeutic index for treatment of ahuman is determined by data with experimental animals from the closestanimal model of the particular type of cancer involved, or from data ontreatment of humans when available. The therapeutic index is defined asthe minimal toxic dose (e.g., the lethal dose for 50% of experimentalanimals) divided by the minimal effective dose (e.g., the minimal doserequired to cause measurable tumor shrinkage in 50% of experimentalanimals).

In other particular embodiments, the CPE or pharmaceutically acceptablesalt thereof has a therapeutic index as administered of at least 2.

In other particular embodiments, the CPE or pharmaceutically acceptablesalt thereof has a therapeutic index as administered of at least 4, atleast 6, at least 8, at least 10, or at least 20.

In some embodiments, the CPE consists of SEQ ID NO:1.

In some embodiments, the CPE is a mutant or engineered CPE. Inparticular embodiments, the mutant or engineered CPE comprises residues45-116 of SEQ ID NO:1.

In some embodiments of the methods of the invention, the method involvesadministering a protective agent that protects cells against CPEtoxicity.

A peptide consisting of residues 290-319 of CPE SEQ ID NO:1 has beenshown to compete with native CPE for binding to cells and thus inhibitCPE binding (26). Any CPE fragment that includes residues 290-319 anddoes not include residues 45-116 is expected to be protective. Such afragment will bind to the CPE receptors (claudin-3 and -4) but not lysethe cells, and will compete with full-length CPE for binding to thereceptor (23-27). Fragments of residues 290-319 of SEQ ID NO:1 are alsoenvisioned. In specific embodiments, the fragment includes residues315-319 of SEQ ID NO:1.

Thus, one protective agent that protects cells against CPE toxicity is aprotective agent that comprises residues 290-319 of native CPE, or ahomologue thereof.

A protective agent that comprises residues 290-319 of native CPE, or ahomologue thereof, is a proteinaceous agent that binds to claudin-3and/or claudin-4 and does not lyse the cells it binds to. In particularembodiments, the homologue of residues 290-319 of native CPE is at least80% or at least 90% identical to residues 290-319 of SEQ ID NO:1.

Another embodiment of a protective agent is a protective agent thatcomprises a fragment of residues 290-319 of SEQ ID NO:1 that bindsspecifically to claudin-3 and/or claudin-4.

The protective agent can also be an antibody against claudin-3 and/orclaudin-4 that inhibits binding of CPE to claudin-3 and/or claudin-4.The term an “antibody against claudin-3 and/or claudin-4” refers to anantibody that specifically binds claudin-3 (i.e., binds claudin-3 anddoes not bind other proteins in a mammal), specifically binds claudin-4,or specifically binds to both claudin-3 and claudin-4.

The protective agent that is an antibody against claudin-3 and/orclaudin-4 can be an antibody comprising an Fc region coupled to apeptide comprising residues 290-319 of SEQ ID NO:1 or a fragment thereofthat specifically binds claudin-3 and/or claudin-4.

In particular embodiments, the protective agent is administeredintravenously and the CPE or pharmaceutically acceptable salt thereof isadministered by a non-intravenous route. For instance, the CPE or saltthereof may be administered intraperitoneally and the protective agentadministered intravenously.

In particular embodiments, the protective agent is administeredenterally. The CPE or salt thereof may be administered in conjunctionwith this, e.g., intraperitoneally or intravenously.

In particular embodiments, the protective agent is a non-toxic agentthat inhibits CPE binding to claudin-3 and/or claudin-4. The inhibitionmay be competitive or non-competitive. Preferably the inhibition iscompetitive.

One embodiment of the invention is a method of treating cancer in amammal involving intraperitoneally administering to the mammal atherapeutically effective amount of CPE or a pharmaceutically acceptablesalt thereof, wherein at least some cancerous cells are located in oradjacent to the peritoneal cavity of the mammal, and the cells aresensitive to CPE. The cancerous cells are considered “adjacent to theperitoneal cavity” of the mammal if intraperitoneal administration ofCPE results in a higher therapeutic index (the ratio of the toxic doseto the effective dose) than systemic intravenous administration of CPE.Cancerous cells may also be considered adjacent to the peritoneal cavityif intraperitoneal administration of CPE results in a higherconcentration of CPE in the cancerous cells than systemic intravenousadministration of CPE.

In a particular embodiment, all detectable cancerous cells are in oradjacent to the peritoneal cavity.

In particular embodiments of the method involving intraperitonealadministration of CPE, the cancerous cells includechemotherapy-resistant cells.

In particular embodiments, the cancerous cells include metastaticcancerous cells.

In particular embodiments, the cancer treated with intraperitonealadministration of CPE is pancreatic cancer. In other embodiments, thecancer is ovarian cancer or uterine cancer.

In particular embodiments, the cancer is uterine serous papillarycarcinoma (USPC).

In another particular embodiment, the cancer is ovarian serous papillarycarcinoma (OSPC).

In particular embodiments, the cancer is liver cancer, stomach cancer,colon cancer, bladder cancer, kidney cancer, intestinal cancer,testicular cancer, or prostate cancer, wherein cells of the cancer haveclaudin-3 and/or claudin-4.

The method involving intraperitoneal administration of CPE may involvein addition administering intravenously or enterally a protective agentthat protects cells against CPE toxicity. This will provide someprotection to healthy non-target tissues.

One embodiment of the invention is a use of CPE to prepare a medicamenteffective to treat ovarian or uterine cancer in a mammal.

In a particular embodiment, the mammal is a human.

In a particular embodiment, the medicament is adapted forintraperitoneal administration to the mammal.

Another embodiment of the invention is a use of CPE to prepare amedicament effective to treat cancer in a mammal, wherein the medicamentis adapted for intraperitoneal administration.

In particular embodiments of the use, the cancer is ovarian cancer,uterine cancer, or pancreatic cancer.

The invention also provides a method of determining the sensitivity of agynecological malignancy to CPE involving detecting the presence orabsence of claudin-3 and/or claudin-4 in a tissue sample comprising aportion of the malignancy.

In particular embodiments, the detecting step involves contacting thetissue sample with a protein containing residues 290-319 of SEQ ID NO:1.In specific embodiments, the protein is CPE. The CPE may be SEQ ID NO:1.In particular embodiments, the protein is an antibody comprisingresidues 290-319 of SEQ ID NO:1 or a fragment thereof that bindsspecifically to claudin-3 and/or claudin-4 (e.g., an antibody comprisingan Fc coupled to an antigen-binding peptide comprising residues 290-319of SEQ ID NO:1).

Binding of the protein can be detected in various ways. If the proteinis CPE, lysis of the cells can be detected. If the protein is labeledwith radioactivity, a fluorescent label, or some other label, the labelcan be detected. The protein can also include a tag that is recognizedby an antibody, where binding of the protein is detected by addingantibody after adding the protein, and detecting binding of the antibodyto the tissue sample.

The detecting step can involve detecting lysis of cells.

The detecting step can involve contacting the tissue sample with anantibody against claudin-3 and/or an antibody against claudin-4.

In Example 1 below it is shown that the TROP-1/Ep-CAM gene isoverexpressed, and the Ep-CAM protein is found, in ovarian serouspapillary carcinoma cells. An anti-Ep-CAM antibody, edrecolomab, hasbeen shown to increase survival in patients harboring stage III coloncancer expressing the Ep-CAM protein (28). Thus, one embodiment of theinvention is a method of treating cancer in a mammal involvingadministering to the mammal a therapeutically effective amount of ananti-Ep-CAM antibody; wherein the cancer is ovarian cancer.

In particular embodiments, the ovarian cancer is OSPC.

In particular embodiments, the antibody is edrecolomab.

The antibody may be radioactive, in which case it can be used forradiation therapy of the cancer.

Another embodiment of the invention is a method of determining thesensitivity of a gynecological malignancy to treatment with ananti-Ep-CAM antibody involving detecting the presence or absence ofEp-CAM in a tissue sample comprising a portion of the malignancy.

The detecting step can include contacting the tissue sample with anantibody against Ep-CAM. The antibody can be polyclonal or monoclonal.

In particular embodiments, the malignancy is ovarian cancer, orspecifically OSPC.

Another embodiment of the invention is a method of protecting a mammalfrom CPE toxicity involving administering a non-toxic protective agentthat binds to claudin-3 and/or claudin-4 and inhibits CPE binding toclaudin-3 and/or claudin-4. The protective agent can be administeredbefore CPE is administered for treatment. Or the protective agent can beadministered after CPE is administered, e.g., as an antidote to anoverdose of therapeutic CPE, or as an antidote or treatment for CPE foodpoisoning.

The protective agent could be an antibody against claudin-3 and/orclaudin-4, e.g., a humanized antibody.

The protective agent can also be an agent that comprises residues290-319 of SEQ ID NO:1, or a homologue thereof, or can be an agent thatcomprises a fragment of residues 290-319 of SEQ ID NO:1 that bindsspecifically to claudin-3 and/or claudin-4.

Another embodiment of the invention provides a method of treating cancerin a mammal involving administering to the mammal an antibody againstclaudin-3 and/or claudin-4, wherein cancerous cells in the mammalcontain claudin-3 and/or claudin-4. Preferably, the cancerous cellscontain at least 5-times more claudin-3 and/or claudin-4 than normalcells of the same tissue type.

In certain embodiments, the antibody may inhibit CPE binding toclaudin-3 and/or claudin-4. But to treat cancer, the antibody does nothave to. It need only bind to the claudin-3 or claudin-4 and attractimmune attack against the antibody-labeled cells.

The antibody may also carry a therapeutic radioisotope, so that it killsthe claudin-3- or claudin-4-containing cells with radioactivity.

In particular embodiments, the cancer treated with antibodies is ovariancancer (e.g., OSPC) or uterine cancer (e.g., USPC). In otherembodiments, the cancer is pancreatic, prostate, or colon cancer. Inother embodiments, the cancer is breast cancer or lung cancer.

Antibodies against claudin-3 and claudin-4 and Ep-CAM can be generatedby injecting rabbits, mice, goats, or other appropriate vertebrateanimals with whole claudin-3 or claudin-4 or Ep-CAM, or peptides fromthe proteins can be synthesized and coupled to a carrier protein forinjection. Preferably, if peptides are used, extracellular domains ofthe proteins are selected, since these are the portions that will beexposed on the exterior of the cell. The amino acid sequences of humanclaudin-4 (referred to as human CPE receptor) and claudin-3 (referred toas RVP1) and hydrophobicity plots of the proteins, which suggest thetransmembrane domains and the extracellular domains, are disclosed inreferences 15 and 50. This can be used to select peptides to be used togenerate antibodies.

Polyclonal or monoclonal antibodies can be prepared from the immunizedanimal as described below. The antibodies used should recognize theexposed extracellular portions of claudin-3 and/or claudin-4 in order tobind to cells expressing the proteins. For this reason, monoclonalantibodies may be preferable, since they can be characterized asrecognizing a specific epitope found on the outer surface of the cellsexpressing claudin-3 or claudin-4.

Antibodies obtained can be administered to experimental mammals at adose of approximately 10 mg/kg every 3-7 days to treat cancer.

Antibodies can be labeled with radioisotopes, such as yttrium-90 oriodine-131 for therapeutic radiation treatment. One procedure forattaching radioisotopes to antibodies is described in reference 122.Radiolabelled antibodies are administered to experimental animals atappropriate radiation doses. For instance, yttrium-90 is administered tohumans in the monoclonal antibody ZEVALIN™ for treatment of lymphoma ata dose of 5 mCi. Thus, in some embodiments of the antibodies used in theinvention, the antibody carries a therapeutic radionuclide.

One embodiment of the invention is an antibody comprising anantigen-binding region comprising residues 290-319 of SEQ ID NO:1 or afragment thereof that binds specifically to claudin-3 and/or claudin-4.This can be created by recombinant means as described in Example 4below. Briefly, a recombinant DNA is engineered to express a fusionpolypeptide comprising the CH2 and CH3 domains of the IgG1 heavy chaincoupled to residues 290-319 of SEQ ID NO:1. Two heavy chains dimerize invivo to form an Fc coupled to two peptides containing the residues ofCPE that bind specifically to claudin-3 and claudin-4.

Thus, in particular embodiments, an antibody used in the invention isproduced by recombinant means.

Other methods for the preparation of antibodies are described below.

Another embodiment of the invention provides a method of treating cancerin a mammal involving administering to the mammal a therapeuticallyeffective amount of a cytotoxic agent that binds specifically toclaudin-3 and/or claudin-4, wherein the cancer is uterine cancer orovarian cancer.

The cytotoxic agent may be CPE or a pharmaceutically acceptable saltthereof.

The cytotoxic agent may also be an antibody that binds specifically toclaudin-3 and/or claudin-4.

The cytotoxic agent may include residues 290-319 of SEQ ID NO:1 or afragment thereof that binds specifically to claudin-3 and/or claudin-4;coupled to a cytotoxic moiety.

In particular embodiments, the cytotoxic moiety is an anti-cancerchemotherapy agent. An anti-cancer chemotherapeutic agent refers to anagent that kills or inhibits the growth of cancer cells while havingless effect on non-cancerous cells, typically because it selectivelykills dividing cells. Examples suitable for use in creating thesecytotoxic agents include amsacrine, azacytidine, bleomycin, busulfan,capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,cladribine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin,decarbazine, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine,hexamethylmelamine, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin C,mitotane, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,pentostatin, plicamycin, procarbazine, ralitrexed, semustine,streptozocin, temozolamide, teniposide, thioguanine, thiotepa,topotecan, trimitrexate, valrubicin, vincristine, vinblastine,vindestine, or vinorelbine.

The chemotherapeutic agents can be coupled to the CPE peptide that bindsclaudin-3/claudin-4 as described in published international patentapplication PCT/US2004/034704.

In other embodiments, the cytotoxic moiety is a moiety that stimulatescell killing by immune system components, e.g., cell killing by thecomplement system or T-cells. In particular embodiments, the cytotoxicmoiety is an antibody Fc. In other specific embodiments, the cytotoxicmoiety is interleukin-2.

It has been shown that interleukin-2 (IL2) can be coupled to an antibodythat recognizes an antigen on cancer cells, and the coupled moiety bindsto target cells through the antibody portion of the conjugate (123). Theinterleukin-2 portion of the conjugate is recognized by T-cells,including T-cells that do not specifically recognize the tumor cells(123). The conjugates have been shown to thereby couple target tumorcells to noncognate T-cells (123). The conjugates also induce cellkilling of the target tumor cells by T-cells, including T-cells that donot specifically recognize the target tumor cells in the absence of theantibody-IL2 conjugate (123).

Thus, here IL2 can be coupled to residues 290-319 of SEQ ID NO:1 or afragment thereof that binds specifically to claudin-3 and/or claudin-4.The fusion protein will bind to cells carrying claudin-3 or claudin-4receptors and induce killing of these cells by cytotoxic T lymphocytesthrough the T-cells' recognition of, binding to, and stimulation by theIL2 moiety.

In another embodiment of this method of treating cancer using acytotoxic agent comprising residues 290-319 of SEQ ID NO:1 or a fragmentthereof that specifically binds claudin-3 and/or claudin-4; coupled to acytotoxic moiety, the cytotoxic moiety is a therapeutic radionuclide.

The methods described herein using agents other than CPE are, like themethods using CPE, suitable for treatment of ovarian cancer and uterinecancer. They are also suitable for treatment of ovarian, uterine, andother cancers wherein the cancer includes chemotherapy resistant cellsor metastatic malignant cells, or wherein the cancer is a recurrentcancer.

Raising Antibodies

To generate antibodies, claudin-3 or claudin-4 or Ep-CAM can beadministered directly to a mammal, or the proteins or peptide fragmentsthereof can be coupled to a carrier protein. Suitable carrier proteinsinclude keyhole limpet hemocyanin, bovine serum albumin, and ovalbumin.Methods of coupling to the carrier protein include single stepglutaraldehyde coupling and other methods disclosed in Harlow, Ed etal., Antibodies: a laboratory manual, Cold Spring Harbor Laboratory(1988).

The immunogen is used to immunize a vertebrate animal in order to inducethe vertebrate to generate antibodies. Preferably the immunogen isinjected along with an adjuvant such as Freund's adjuvant, to enhancethe immune response. Suitable vertebrates include rabbits, mice, rats,hamsters, goats, and chickens.

Hybridomas to synthesize monoclonal antibodies can be prepared bymethods known in the art. See, for instance, Wang, H., et al., AntibodyExpression and Engineering, Am. Chem. Soc., Washington, D.C. (1995).Polyclonal and monoclonal antibodies can be isolated by methods known inthe art. See, for instance, id. and Harlow et al.

Native antibodies are tetramers of two identical light (L) chains andtwo identical heavy (H) chains. The L and H chains each have variabledomains that are responsible for antigen recognition and binding. Thevariability in the variable domains is concentrated in thecomplementarity determining regions (CDRs).

An antibody that is contemplated for use in the present invention can bein any of a variety of forms, including a whole immunoglobulin, anantibody fragment such as Fv, Fab, and similar fragments, a single chainantibody that includes the CDR, and like forms, all of which fall underthe broad term “antibody” as used herein.

The term “antibody fragment” refers to an antigen-binding portion of afull-length antibody. Antibody fragments can be as small as about 4amino acids, about 10 amino acids, or about 30 amino acids or more. Sometypes of antibody fragments are the following:

(1) Fab is the fragment that contains a monovalent antigen-bindingfragment of an antibody molecule. A Fab fragment can be produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain. Two Fab fragments areobtained per whole antibody molecule.

(2) Fab′ is the fragment of an antibody that can be obtained by treatingwhole antibody with pepsin, followed by reduction to yield an intactlight chain and a portion of the heavy chain. Two Fab′ fragments areobtained per whole antibody molecule. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain including one or more cysteines.

(3) F(ab′)₂ is the fragment that can be obtained by digestion of wholeantibody with pepsin, without reduction. F(ab′)₂ is a dimer of two Fab′fragments held together by two disulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. Fv consists of a dimer of one H and one Lchain variable domain in a tight, non-covalent association (V_(H)-V_(L)dimer). It is in this configuration that the three CDRs of each variabledomain interact to define an antigen-binding site. Collectively, the sixCDRs confer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to bind antigen, although at alower affinity than the complete binding site.

(5) A single chain antibody (SCA) is defined as a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

The preparation of polyclonal antibodies is well known to those skilledin the art. See, for example, Coligan et al., in Current Protocols inImmunology, section 2.4.1 (1992). The preparation of monoclonalantibodies is likewise conventional. See, for example, Harlow et al.,page 726.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature 256:495 (1975), or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clarkson et al., Nature352:624 (1991), as well as in Marks et al., J. Mol. Biol. 222:581(1991). Another method involves humanizing a monoclonal antibody byrecombinant means to generate antibodies containing human specific andrecognizable sequences. See, for review, Holmes et al., J. Immunol.158:2192 (1997) and Vaswani et al., Annals Allergy, Asthma & Immunol.81:105 (1998).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) are identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Nat'l. Acad. Sci. 81:6851 (1984)).

Methods of making antibody fragments are also known in the art (see, forexample, Harlow and Lane, Antibodies: a Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988)). Antibody fragments of the presentinvention can be prepared by proteolytic hydrolysis of the antibody orby expression in E. coli of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)₂. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5 S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab fragments and an Fcfragment directly. These methods are described, for example, in U.S.Pat. Nos. 4,036,945, and 4,331,647, and references contained therein.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker bridging the two V domains. Methods for producing sFvs aredescribed, for example, by Whitlow et al., Methods: a Companion toMethods in Enzymology, 2:97 (1991); Bird et al., Science 242:423 (1988);Ladner et al., U.S. Pat. No. 4,946,778; and Pack et al., Bio/Technology11:1271 (1993).

Another form of an antibody fragment is a peptide containing a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example,Larrick et al., Methods: a Companion to Methods in Enzymology, 2:106(1991).

The invention contemplates human and humanized forms of non-human (e.g.,murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., Nature 321:522(1986); Reichmann et al., Nature 332:323 (1988); Presta, Curr. OpinionStruct. Biol. 2:593 (1992); Holmes et al., J. Immunol. 158:2192 (1997);and Vaswani et al., Annals Allergy, Asthma & Immunol. 81:105 (1998).

Antibodies of the invention can also be mutated to optimize theiraffinity, selectivity, binding strength or other desirable property. Onemethod of mutating antibodies involves affinity maturation using phagedisplay. Affinity maturation using phage display refers to a processdescribed in Lowman et al., Biochemistry 30:10832 (1991); see alsoHawkins et al., J. Mol. Biol. 254:889 (1992).

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES

The following materials and methods are common to the Examples.

Materials and Methods

RNA Purification and Microarray Hybridization and Analysis. Detailedprotocols for RNA purification, cDNA synthesis, cRNA preparation, andhybridization to the Affymetrix Human U95Av2 GeneChip microarray wereperformed according to the manufacturer's protocols, as reportedpreviously (29).

Data Processing. All data used in the analyses were derived fromAffymetrix 5.0 software. GeneChip 5.0 output files are given as a signalthat represents the difference between the intensities of thesequence-specific perfect match probe set and the mismatch probe set, oras a detection of present, marginal, or absent signals as determined bythe GeneChip 5.0 algorithm. Gene arrays were scaled to an average signalof 1500 and then analyzed independently. Signal calls were transformedby the log base 2 and each sample was normalized to give a mean of 0 andvariance of 1.

Gene Expression Data Analysis. Statistical analyses of the data wereperformed with the software package SPSS10.0 (SPSS, Chicago, Ill.). Thefirst test applied was the detection. In each comparison, genes having“present” detection calls in more than half of the samples in theoverexpressed gene group were retained. To compare gene expressionlevels, the nonparametric Wilcoxon rank sum (WRS) test (p<0.05) wasapplied to the normalized signal call. By combining the detection andWRS data, differentially expressed genes were identified between OSPCand NOVA or USPC and NEC.

Quantitative Real-time PCR. q-RT-PCR was performed with an ABI Prism7000 Sequence Analyzer using the manufacturer's recommended protocol(Applied Biosystems, Foster City, Calif.) to validate differentialexpression of selected genes in samples from all primary cell lines (10OSPC and 5 NOVA in Example 1; 10 USPC and 5 NEC in Example 2). Eachreaction was run in triplicate. The comparative threshold cycle (C_(T))method was used for the calculation of amplification fold as specifiedby the manufacturer. Briefly, five μg of total RNA from each sample wasreverse transcribed using SUPERSCRIPT™ II Rnase H Reverse Transcriptase(Invitrogen, Carlsbad, Calif.). Ten μl of reverse transcribed RNAsamples (from 500 μl of total volume) were amplified by using theTAQMAN™ Universal PCR Master Mix (Applied Biosystems) to produce PCRproducts specific for the target genes. Primers specific for 18 sribosomal RNA and empirically determined ratios of 18 s competimers(Applied Biosystems) were used to control for the amounts of cDNAgenerated from each sample. Primers for TROP-1, claudin-3 and claudin-4were obtained from Applied Biosystems as assay on demand products.Assays ID were Hs00158980_m1 (TROP-1), Hs00265816_s1 (claudin-3), andHs00533616_s1 (claudin-4). CD24 primers sequences were the following(forward, 5′-cccaggtgttactgtaattcctcaa; reverse,5′-gaacagcaatagctcaacaatgtaaac). Primers for L1CAM, claudin-3, andclaudin-4 were obtained from Applied Biosystems as assay on demandproducts. Assays ID were Hs00170849_m1 (L1CAM,), Hs00265816_s1(claudin-3), and Hs00533616_s1 (claudin-4). GRB7 primer sequences were:forward, 5′-tctacgggatgaccactga-3′; reverse, 5′-cgaagccccttgtgtcca-3′.c-erbB2 primer sequences were: forward, 5′-gtatacattcggcgccagct-3′;reverse, 5′-gcagacgagggtgcagga-3′. CDKN2A/p16 primer sequences were:forward, 5′-cccaaacgcaccgaatagttac-3′; reverse,5′-attccaattcccctgcaaact-3′. CDKN2A/p14ARF primer sequences were:forward, 5′-tgatgctactgaggagccagc-3′; reverse,5′-agggcctttcctacctggtc-3′. Amplification was carried out by using 1unit of polymerase in a final volume of 20 μl containing 2.5 mM MgCl₂.TAQGOLD™ was activated by incubation at 96° C. for 12 min, and thereactions were cycled 26-30 times at 95° C. for 1 min, 55° C. for 1 min,and 72° C. for 1 min, followed by a final extension at 72° C. for 10min. PCR products were visualized on 2% agarose gels stained withethidium bromide, and images were captured by an Ultraviolet ProductsImage Analysis System. Differences among OSPC and NOVA or USPC and NECin the q-RT-PCR expression data were tested using the Kruskal-Wallisnonparametric test. Pearson product-moment correlations were used toestimate the degree of association between the microarray and q-RT-PCRdata.

Claudin-4 Immunostaining of Formalin-fixed Tumor Tissues. Ovarian anduterine tumors were evaluated by immunohistochemical staining onformalin-fixed tumor tissue for claudin-4 surface expression. Studyblocks were selected after histopathologic review by a surgicalpathologist. The most representative hematoxylin and eosin-stained blocksections were used for each specimen. Briefly, immunohistochemicalstains were performed on 4 μm-thick sections of formalin-fixed, paraffinembedded tissue. After pretreatment with 10 mM citrate buffer at pH 6.0using a steamer, they were incubated with mouse anti-claudin-4antibodies (Zymed Laboratories Inc. San Francisco, Calif.) at 1:2000dilution. Antigen-bound primary antibodies were detected using standardavidin-biotin immunoperoxidase complex (Dako Corp., Carpinteria,Calif.). Cases with less than 10% staining in tumor cells wereconsidered negative for claudin expression, while positive cases wereclassified as follows regarding the intensity of claudin-4 proteinexpression: (a) +, focal membrane staining; (b) ++, diffuse membranestaining; and c) +++, diffuse membrane and cytoplasmic staining.

Example 1

Gene Expression Profiles in Primary Ovarian Serous Papillary Tumors andNormal Ovarian Epithelium: Identification of Candidate Molecular Markersfor Ovarian Cancer Diagnosis and Therapy

The goal of this study was to identify genes with differential patternsof expression between ovarian serous papillary carcinoma (OSPC) andnormal ovarian (NOVA) epithelium and use this knowledge for thedevelopment of novel diagnostic and therapeutic markers for ovariancancer. Gene expression in 10 primary OSPC cell lines, 2 establish OSPCTcell lines (UCI-101, UCI-107), and 5 primary NOVA epithelial cultureswas analyzed by oligonucleotide microarrays with probe setscomplementary to 12,533 genes.

Materials and Methods

Establishment of OSPC and NOVA Primary Cell Lines. A total of fifteenprimary cell lines (i.e., 10 OSPC and 5 NOVA) were established aftersterile processing of the samples from surgical biopsies as previouslydescribed for ovarian carcinoma specimens (30,19,31). UCI-101 andUCI-107, two previously characterized OSPC cell lines (32,33) were alsoincluded in the analysis. All fresh tumor samples were obtained withappropriate consent according to IRB guidelines. Tumors were stagedaccording to the F.I.G.O. operative staging system. Radical tumordebulking, including a total abdominal hysterectomy and omentectomy, wasperformed in all ovarian carcinoma patients while normal ovarian tissuewas obtained from consenting similar age donors undergoing surgery forbenign pathology. No patient received chemotherapy before surgery.Patient characteristics are described in Table 1. Briefly, normal tissuewas obtained by scraping epithelial cells from the ovarian surface andplacing cells in RPMI 1640 medium (Sigma Chemical Co., St. Louis, Mo.)containing 10% fetal bovine serum (FBS, Invitrogen, Grand Island, N.Y.),200 u/ml penicillin, and 200 μg/ml streptomycin. The epithelial explantswere then allowed to attach and proliferate. Once the epithelial cellsreached confluency, explants were trypsinized and subcultured for 3 to 4passages before being collected for RNA extraction. Viable tumor tissuewas mechanically minced in RPMI 1640 to portions no larger than 1-3 mm³and washed twice with RPMI 1640. The portions of minced tumor were thenplaced into 250 ml flasks containing 30 ml of enzyme solution [0.14%collagenase Type I (Sigma, St. Louis, Mo.) and 0.01% DNAse (Sigma, 2000KU/mg)] in RPMI 1640, and incubated on a magnetic stirring apparatusovernight at 4° C. Enzymatically dissociated tumor was then filteredthrough 150 μm nylon mesh to generate a single-cell suspension. Theresultant cell suspension was then washed twice in RPMI 1640 plus 10%FBS. Primary cell lines were maintained in RPMI 1640, supplemented with10% FBS, 200 u/ml penicillin, 200 μg/ml streptomycin at 37° C., 5% CO₂.The epithelial nature and the purity of OSPC and NOVA cultures wasverified by immunohistochemical staining and flow cytometric analysiswith antibodies against cytokeratin as previously described (30,31).Only primary cultures which had at least 90% viability andcontained >99% epithelial cells were used for total RNA extraction.

Gene Cluster/Treeview. The hierarchical clustering of average-linkagemethod with the centered correlation metric was used (34). Thedendrogram was constructed with a subset of genes from 12,588 probe setspresent on the microarray, whose expression levels varied the most amongthe 11 samples, and were thus most informative. For the hierarchicalclustering shown in FIGS. 1 and 2, only genes significantly expressedand whose average change in expression level was at least five-fold werechosen. The expression value of each selected gene was re-normalized tohave a mean of zero.

Flow Cytometry. To validate microarray data on primary OSPC and NOVAcell lines at the protein level, TROP-1/EP-CAM and CD24 expression wereevaluated by flow cytometric analysis of a total of 13 primary celllines (i.e., 10 OSPC and 3 NOVA). Unconjugated anti-TROP-1/EP-CAM(IgG2a), anti-CD24 (IgG2a) and isotype control antibodies (mouse IgG2a)were all obtained from BD PharMingen (San Diego, Calif.). Goatanti-murine FITC-labeled mouse Ig was purchased from Becton Dickinson(San Jose, Calif.). Analysis was conducted with a FACScan, utilizingcell Quest software (Becton Dickinson).

TROP-1 and CD24 Immunostaining of Formalin-fixed Tumor Tissues. Toevaluate whether the differential TROP-1 and CD24 expression detected byflow cytometry on primary OSPC cell lines were comparable to theexpression of TROP-1 and CD24 of uncultured OSPC from which the primarycell lines were derived, protein expression was evaluated by standardimmunohistochemical staining on formalin fixed tumor tissue from allsurgical specimens (i.e., 10 OSPC and 5 NOVA controls). Study blockswere selected after histopathologic review by a surgical pathologist.The most representative hematoxylin and eosin-stained block sectionswere used for each specimen. Briefly, immunohistochemical stains wereperformed on 4 μm-thick sections of formalin-fixed, paraffin embeddedtissue. After pretreatment with 10 mM citrate buffer at pH 6.0 using asteamer, they were incubated with anti-Ep-CAM Ab-3 MAb and anti-CD24(Neo Markers, Fremont, Calif.) at 1:2000 dilution. Slides weresubsequently labeled with streptavidin-biotin (DAKO, Glostrup, Denmark),stained with diaminobenzidine and counterstained with hematoxylin. Theintensity of staining was graded as 0 (staining not greater thannegative control), 1+ (light staining), 2+ (moderate staining), or 3+(heavy staining).

Results

Characteristics of the Patients. The characteristics of the patientsfrom whom the 10 primary OSPC cell lines were derived are listed inTable 1.

TABLE 1 Presence of Chemotherapy Response to Patient Age Grade StageAscites Regimen Therapy OSPC 1 42 G2/3 IV A yes TAX + CARB Completeresponse OSPC 2 67 G3 III B yes TAX + CARB Complete response OSPC 3 61G3 III C no TAX + CARB Partial response OSPC 4 60 G3 III C no TAX + CARBComplete response OSPC 5 59 G2/3 III C yes TAX + CARB Complete responseOSPC 6 72 G3 IV A yes TAX + CARB Stable disease OSPC 7 63 G3 III C yesTAX + CARB Progressive Disease OSPC 8 74 G2/3 III C no TAX + CARBPartial response OSPC 9 68 G3 III B yes TAX + CARB Complete responseOSPC 10 77 G2/3 III C no TAX + CARB Complete response

Gene Expression Profiles Distinguish OSPC From NOVA and IdentifyDifferentially Expressed Genes. Flash frozen biopsies from ovarian tumortissue are known to contain significant numbers of contaminant stromalcells as well as a variety of host derived immune cells (e.g.,monocytes, dendritic cells, lymphocytes). In addition, because ovarianepithelial cells represent a small proportion of the total cells foundin the normal ovary, it is difficult to collect primary material that isfree of contaminating ovarian stromal cells in sufficient quantities toconduct comparative gene expression analyses. However, ovarianepithelial cells can be isolated and expanded in culture for about 15passages (30,19) while the majority of primary ovarian carcinomas can beexpanded in vitro for several passages (31). Thus, short term primaryOSPC and NOVA cell cultures, minimizing the risk of a selection biasinherent in any long term in vitro growth, may provide an opportunity tostudy differential gene expression between relatively pure populationsof normal and tumor-derived epithelial cells. Accordingly, comprehensivegene expression profiles of 10 primary OSPC and 5 primary NOVA celllines were generated using high-density oligonucleotide arrays with12,533 probe sets, which in total interrogated some 10,000 genes. Inaddition, gene expression profiles derived from two established andpreviously characterized OSPC cell lines (i.e., UCI-101 and UCI-107)were also analyzed. By combining the detection levels of genessignificantly expressed in primary and established OSPC cultures verylittle correlation between the two groups of OSPC was found. Indeed, asshown in FIG. 1, UCI-101 and UCI-107 established cell lines groupedtogether in the leftmost columns of the dendrogram while all ten primaryOSPC clustered tightly together in the rightmost columns separately bythe 5 NOVA controls. Because of these results the analysis was focusedon the detection of differentially expressed genes between the twohomogeneous groups of primary OSPC and NOVA cell lines. Using thenonparametric WRS test (p<0.05) that readily distinguished between thetwo groups of primary cultures, 1,518 genes differentially expressedbetween OSPC and NOVA were found. The cluster analysis was based onhybridization intensity values for each cell line for 299 gene segmentswhose average change in expression level was at least five-fold (datanot shown). All 10 OSPC were grouped together. Similarly, all 5 NOVAwere found to cluster tightly together. The tight clustering of OSPCfrom NOVA was “driven” by two distinct profiles of gene expression. Thefirst was represented by a group of 129 genes that were highly expressedin OSPC and underexpressed in NOVA (Table 2). Many genes shownpreviously to be involved in ovarian carcinogenesis are present on theselists, providing some validity to the array analysis, while others arenovel in ovarian carcinogenesis. Included in the genes overexpressed inOSPC are laminin, claudin-3 (CLDN3) and claudin-4 (CLDN4),tumor-associated calcium signal transducer 1 and 2 (TROP-1/Ep-CAM,TROP-2), ladinin 1, S100A2, SERPIN2 (PAI-2), CD24, lipocalin 2,osteopontin, kallikrein 6 (protease M) and kallikrein 10, matriptase(TADG-15) and stratifin (Table 2). Importantly, TROP-1/Ep-CAM geneencoding for a transmembrane glycoprotein previously found to beoverexpressed in various carcinoma types including colorectal and breast(35) and where antibody-directed therapy has resulted in improvedsurvival of patients (28), was 39-fold differentially expressed in OSPCwhen compared to NOVA (Table 2). The second profile was represented by170 genes that were highly expressed in NOVA and underexpressed in OSPC(data not shown). Included in this latter group of genes aretransforming growth factor beta receptor III, platelet-derived growthfactor receptor alpha, SEMACAP3, ras homolog gene family, member I(ARHI), thrombospondin 2 and disabled-2/differentially expressed inovarian carcinoma 2 (Dab2/DOC2).

TABLE 2 Upregulated genes expressed at least 5 fold higher in OSPCcompared with NOVA Ratio Probe Set Gene Symbol Score(d)(SAM) p of WRSOva/Nova 35280_at LAMC2 1.68927386 0.006 46.45 35276_at CLDN41.734410451 0.015 43.76 33904_at CLDN3 1.650076713 0.02 40.24 575_s_atTACSTD1 1.705816336 0.02 39.36 32154_at TFAP2A 1.667038647 0.002 33.3139015_f_at KRT6E 1.062629117 0.047 28.02 1713_s_at CDKN2A 1.1376829050.015 26.96 41376_i_at UGT2B7 0.939735032 0.047 24.81 38551_at L1CAM1.151935363 0.008 24.66 291_s_at TACSTD2 1.249487388 0.047 24.4633282_at LAD1 1.422481563 0.006 24.31 34213_at KIBRA 1.533570321 0.00223.06 38489_at HBP17 1.522882814 0.004 22.54 36869_at PAX8 1.439068360.004 22.20 38482_at CLDN7 1.307716566 0.027 20.01 37909_at LAMA31.121654521 0.027 19.24 34674_at S100A1 1.219106334 0.008 19.01 1620_atCDH6 0.908193479 0.036 18.69 32821_at LCN2 1.99990601 0.008 18.13522_s_at FOLR3 1.113781518 0.02 17.90 39660_at DEFB1 0.837612681 0.03617.34 2011_s_at BIK 1.594057668 0.006 17.23 41587_g_at FGF18 0.9657269830.02 17.10 36929_at LAMB3 1.115590892 0.047 16.76 35726_at S100A21.036576352 0.004 15.05 1887_g_at WNT7A 1.186990893 0.004 14.75 35879_atGAL 1.223278825 0.002 14.65 266_s_at CD24 1.756569076 0.004 14.451108_s_at EPHA1 1.242309171 0.006 14.36 37483_at HDAC9 1.406744957 0.00614.28 31887_at — 1.311220827 0.011 13.68 1788_s_at DUSP4 1.224219870.003 13.65 32787_at ERBB3 0.996784565 0.02 13.21 41660_at CELSR11.634286803 0.004 13.11 33483_at NMU 1.100849065 0.004 13.04 31792_atANXA3 0.896090153 0.011 12.90 36838_at KLK10 1.026306829 0.02 12.711585_at ERBB3 1.102058608 0.011 12.51 1898_at TRIM29 1.071987353 0.00212.44 37185_at SERPINB2 0.815945986 0.027 12.26 406_at ITGB4 1.2961945590.006 11.66 1914_at CCNA1 0.936342778 0.011 11.21 977_s_at CDH10.93637461 0.036 11.19 37603_at IL1RN 1.103624942 0.015 11.14 35977_atDKK1 1.123240701 0.006 10.74 36133_at DSP 1.280269127 0.002 10.6936133_s_at TNNT1 1.269558595 0.002 10.19 1802_s_at ERBB2 0.7874657060.006 9.61 2092_s_at SPP1 1.34315986 0.02 9.53 35699_at BUB1B1.026388835 0.006 9.49 37554_at KLK6 0.895036336 0.027 9.45 38515_atBMP7 0.945367 0.027 9.32 34775_at TSPAN-1 1.001195829 0.02 9.01 37558_atIMP-3 1.023799379 0.011 8.99 38324_at LISCH7 1.308000521 0.006 8.9639610_at HOXB2 1.355268631 0.006 8.64 572_at TTK 1.122796615 0.006 8.531970_s_at FGFR2 1.022708001 0.02 8.30 160025_at TGFA 1.065272755 0.0158.28 41812_s_at NUP210 1.392787031 0.006 8.26 34282_at NFE2L31.165273649 0.008 8.06 2017_s_at CCND1 1.114984456 0.002 8.04 33323_r_atSFN 1.202433185 0.008 8.01 38766_at SRCAP 1.131917941 0.008 7.9941060_at CCNE1 1.151246634 0.006 7.97 39016_r_at KRT6E 0.973486831 0.0087.91 31610_at MAP17 1.0156502 0.027 7.81 2027_at S100A2 0.9419190010.008 7.76 418_at MKI67 0.826426448 0.011 7.46 1536_at CDC6 1.088689410.017 7.37 634_at PRSS8 0.899891713 0.02 7.30 34342_s_at SPP11.318723271 0.02 7.27 182_at ITPR3 1.107167336 0.006 7.27 32382_at UPK1B0.731294678 0.047 7.16 863_g_at SERPINB5 0.783530451 0.015 7.14 904_s_atTOP2A 0.971648429 0.02 7.12 40095_at CA2 0.798857154 0.027 7.02 41294_atKRT7 1.082553892 0.011 7.00 39951_at PLS1 0.995091449 0.006 6.9438051_at MAL 0.819842532 0.036 6.82 40726_at KIF11 0.803689697 0.0366.78 1148_s_at — 0.683569558 0.047 6.72 37920_at PITX1 0.996497645 0.0156.67 37117_at ARHGAP8 1.129131077 0.002 6.65 38881_i_at TRIM160.721698355 0.047 6.59 34251_at HOXB5 1.219463307 0.002 6.52 41359_atPKP3 1.047269618 0.004 6.50 40145_at TOP2A 0.961173129 0.02 6.4837534_at CXADR 0.888147605 0.006 6.32 40303_at TFAP2C 0.948734146 0.0046.30 31805_at FGFR3 0.969764101 0.011 6.28 33245_at MAPK13 0.8775145860.011 6.27 885_g_at ITGA3 0.702747685 0.036 6.19 34693_at STHM0.872525584 0.008 6.15 38555_at DUSP10 0.880305317 0.008 6.12 38418_atCCND1 1.071102249 0.002 5.97 33730_at RAI3 0.813298748 0.011 5.9039109_at TPX2 1.040973216 0.011 5.87 36658_at DHCR24 1.122129795 0.0045.81 35281_at LAMC2 0.747766326 0.047 5.78 38749_at MGC29643 0.6832750860.036 5.77 1083_s_at MUC1 0.746980491 0.027 5.75 40079_at RAI30.709840659 0.02 5.73 2047_s_at JUP 0.815282235 0.011 5.62 32275_at SLP10.940625784 0.02 5.61 2020_at CCND1 0.926408163 0.002 5.51 33324_s_atCDC2 1.026683994 0.008 5.47 36863_at HMMR 0.96343264 0.006 5.46 1657_atPTPRR 0.764510362 0.02 5.41 37985_at LMNB1 0.895475347 0.008 5.3636497_at C14orf78 0.942921564 0.008 5.33 2021_s_at CCNE1 0.8932282970.006 5.33 37890_at CD47 0.775908217 0.015 5.33 40799_at C16orf340.852774782 0.008 5.30 35309_at ST14 0.852534105 0.008 5.30 1599_atCDKN3 0.925527261 0.02 5.29 981_at MCM4 1.058558782 0.006 5.28 32715_atVAMP8 0.938171642 0.006 5.28 38631_at TNFAIP2 0.72369235 0.015 5.2634715_at FOXM1 1.31035831 0.008 5.24 33448_at SPINT1 0.924028022 0.0155.21 419_at MKI67 0.938133197 0.015 5.16 1651_at UBE2C 1.436239741 0.0085.14 35769_at GPR56 0.937347548 0.015 5.08 37310_at PLAU 0.8851107410.036 5.08 36761_at ZNF339 0.937123503 0.011 5.05 37343_at ITPR31.001079303 0.003 5.05 40425_at EFNA1 0.813414458 0.047 5.04 1803_atCDC2 0.732852195 0.027 5.00

Validation of the Microarray Data. Quantitative RT-PCR assays were usedto validate the microarray data. Four highly differentially expressedgenes between OSPC and NOVA (i.e., TROP-1, CD24, Claudin-3 andClaudin-4) were selected for q-RT-PCR analysis. A comparison of themicroarray and q-RT-PCR data for these genes is shown in FIG. 2.Expression differences between OSPC and NOVA for TROP-1, (p=0.002), CD24(p=0.03), claudin-3 (p=0.002) claudin-4 (p=0.001) were readily apparent(Table 2 and FIG. 2). Moreover, for all four genes tested, the q-RT-PCRdata were highly correlated to the microarray data (p<0.001) (r=0.90,0.82. 0.80 and 0.75, respectively), as estimated from the samples (i.e.,10 OSPC and 5 NOVA) included in both the q-RT-PCR and microarrayexperiments. The q-RT-PCR data mirror the microarray data, bothqualitatively and quantitatively, and suggest that most array probe setsare likely to accurately measure the levels of the intended transcriptwithin a complex mixture of transcripts.

TROP-1 and CD24 Expression by Flow Cytometry on Primary OSPC and NOVACell Lines. An important issue is whether differences in gene expressionresult in meaningful differences in protein expression. BecauseTROP-1/Ep-CAM gene encodes the target for the anti-Ep-CAM antibody(17-1A) Edrecolomab (Panorex) that has previously been shown to increasesurvival in patients harboring stage III colon cancer (28), expressionof Ep-CAM protein by FACS analysis was analyzed on 13 primary cell lines(i.e., 10 OSPC and 3 NOVA). As positive controls, breast cancer celllines (i.e., BT-474 and SK-BR-3, American Type Culture Collection) knownto overexpress TROP-1/Ep-CAM were also studied. High TROP-1/Ep-CAMexpression was found on all three primary OSPC cell lines tested (100%positive cells for all three OSPC), with mean fluorescence intensity(MFI) ranging from 116 to 280 (FIG. 3). In contrast, primary NOVA celllines were negative for TROP-1/Ep-CAM surface expression (p<0.001) (FIG.3). Similarly, CD24 expression was found on all three primary OSPC celllines tested (100% positive cells for all three OSPC), with meanfluorescence intensity (MFI) ranging from 26 to 55 (FIG. 3). Incontrast, primary NOVA cell lines were negative for CD24 surfaceexpression (p<0.005) (FIG. 3). These results show that high expressionof the TROP-1/Ep-CAM and CD24 gene products by OSPC correlate tightlywith high protein expression by the tumor cells. Breast cancer positivecontrols were found to express high levels of TROP-1/Ep-CAM (data notshown).

TROP-1/Ep-CAM and CD24 Expression by Immunohistology on OSPC and NOVATissue Blocks. To determine whether the high (OSPC) or low (NOVA)expression of the genes and Ep-CAM and CD24 protein expression detectedby microarray and flow cytometry, respectively, in primary cell lines isthe result of a selection of a subpopulation of cancer cells present inthe original tumor, or whether in vitro expansion conditions may havemodified gene expression, immunohistochemical analysis of TROP-1/Ep-CAMand CD24 protein expression on formalin fixed tumor tissue from alluncultured primary surgical specimens of OSPC and NOVA was performed.Heavy apical membranous staining for CD24 protein expression was notedin all OSPC specimens that also overexpressed the CD24 gene and its geneproduct by microarray and flow cytometry, respectively. In contrast,negative or low (i.e., score 0 or 1+) staining was found in all NOVAsamples tested by immunohistochemistry. Similarly, heavy membranousstaining for TROP-1/Ep-CAM protein expression (i.e., score 3+) was notedin all OSPC specimens that also overexpressed the TROP-1/Ep-CAM gene andits gene product by microarray and flow cytometry, respectively. Incontrast, negative or low (i.e., score 0 or 1+) staining was found inall NOVA samples tested by immunohistochemistry (data not shown).

Discussion

Because of the lack of an effective ovarian cancer screening program andthe common development of chemotherapy resistant disease after aninitial response to cytotoxic agents (e.g., a platinum based regimen),ovarian cancer remains the most lethal among the gynecologicmalignancies. Thus, identification of novel ovarian tumor markers to beused for early detection of the disease as well as the development ofeffective therapy against chemotherapy resistant/recurrent ovariancancer remains a high priority.

High-throughput technologies for assaying gene expression, such ashigh-density oligonucleotide and cDNA microarrays, may offer thepotential to identify clinically relevant genes highly differentiallyexpressed between ovarian tumors and normal control ovarian epithelialcells (19,30,36,37,38,39,40,41). This report represents thecommunication of an investigation involving the genome-wide examinationof differences in gene expression between primary OSPC and normalovarian epithelial cells (NOVA). In this study short-term primary OSPCand NOVA cultures were used (to minimize the risk of a selection biasinherent in any long term in vitro growth) to study differential geneexpression in highly enriched populations of epithelial tumor cells. Inthis work, only the cancer cells derived from papillary serous histologytumors, which is the most common histological type of ovarian cancer,were included to limit the complexity of gene expression analysis.

It was found that hierarchical clustering of the samples and geneexpression levels within the samples led to the unambiguous separationof OSPC from NOVA. Of interest, the expression patterns detected inprimary OSPC cells were consistently different from those seen inestablished serous papillary ovarian carcinoma cell lines (i.e., UCI-101and UCI-107). These data, thus, further highlight the divergence of geneexpression that occurs as the result of long-term in vitro growth.Furthermore, these data emphasize that, although established ovariancancer cell lines provide a relatively simple model to examine geneexpression, primary OSPC and NOVA cultures represent a better modelsystem of normal and cancerous ovarian tissues in comparative geneexpression analysis. OVA 2, an OSPC with mixed clear cell features(i.e., a biologically aggressive variant of ovarian cancer characterizedby a poor prognosis) clustered on a sub-branch with OSPC. Because ofthese results, the decision was made to focus the analysis on thedetection of differentially expressed genes between the two homogeneousgroups of primary OSPC and NOVA.

299 genes differentially expressed between OSPC and NOVA whose averagechange in expression level between the two groups was at least five-foldwere detected. The known function of some of these genes may provideinsights in the biology of serous ovarian tumors while others may proveto be useful diagnostic and therapeutic markers against OSPC. Forexample, laminin gamma 2 gene was found to be the most highlydifferentially expressed gene in OSPC with over 46-fold up-regulationrelative to NOVA. Cell migration of ovarian epithelial cells isconsidered essential for cell dissemination and invasion of thesubmesothelial extracellular matrix commonly seen in ovarian cancer.Consistent with this view, the laminin gamma 2 isoform has beenpreviously suggested to play an important role in tumor cell adhesion,migration, and scattering of ovarian carcinoma cells (42,43,44). Thus,it is likely that the high laminin expression found in ovarian tumorcells may be a marker correlated with the invasive potential of OSPC.Consistent with this view, increased cell surface expression of lamininhas been reported in highly metastatic tumors cells compared to cells oflow metastatic potential (45). Importantly, previous work has also shownthat attachment and metastases of tumor cells can be inhibited byincubation with antilaminin antibodies (46), or synthetic lamininpeptides (47), underscoring a novel potential approach for the treatmentof chemotherapy resistant ovarian cancer.

TROP-1/Ep-CAM (also called 17-1A, ESA, EGP40) is a 40 kDa epithelialtransmemebrane glycoprotein found overexpressed in several normalepithelia and in various carcinomas including colorectal and breastcancer (35). In most adult epithelial tissues, enhanced expression ofEp-CAM is closely associated with either benign or malignantproliferation. Among mammals, Ep-CAM is an evolutionarily highlyconserved molecule (48), suggesting an important biologic function ofthis molecule in epithelial cells and tissue. In this regard, Ep-CAM isknown to function as an intercellular adhesion molecule and could have arole in tumor metastasis (49). Because a randomized phase II trial withMAb CO17-1A in colorectal carcinoma patients has demonstrated asignificant decrease in recurrence and mortality of MAb-treated patientsversus control patients (28), TROP-1/Ep-CAM antigen has attractedsubstantial attention as a target for immunotherapy for treating humancarcinomas. Importantly, in this work TROP-1/Ep-CAM was found 39-foldoverexpressed in OSPC when compared to NOVA. These data provide supportfor the notion that anti-Ep-CAM antibody therapy can be a novel, andeffective, treatment option for OSPC patients with residual/resistantdisease after surgical and cytotoxic therapy. Protein expression dataobtained by flow cytometry on primary OPSC cell lines and byimmunohistochemistry on uncultured OSPC blocks support this view.

Claudin-3 and claudin-4, two members of claudin family of tight junctionproteins, were two of the top five differentially expressed genes inOSPC. These results are consistent with a previous report on geneexpression in ovarian cancer (19). Although the function of claudinproteins in ovarian cancer is still unclear, these proteins likelyrepresents a transmembrane receptor (50). Claudin-3 and claudin-4 arelow- and high-affinity receptors, respectively, for CPE and aresufficient to mediate CPE binding and trigger subsequent toxin-mediatedcytolysis (51). These known functions of claudin-3 and claudin-4,combined with their extremely high level of expression in OSPC suggestthe use of CPE as a novel therapeutic strategy for the treatment ofchemotherapy resistant disease in ovarian cancer patients.

Plasminogen activator inhibitor-2 (PAI-2), a gene whose expression hasbeen linked to cell invasion in several human malignancies (52,53), aswell as to protection from tumor necrosis factor-α (TNF-α)-mediatedapoptosis (54) was found 12-fold differentially expressed in OSPC whencompared to NOVA. High PAI-2 levels are independently predictive of apoor disease-free survival (55). Interestingly, a 7-fold increase inPAI-2 content was found in the omentum of ovarian cancer patientscompared to the primary disease suggesting that metastatic tumors mayoverexpress PAI-2 (55). Other studies, however, have identified PAI-2production as a favorable prognostic factor in epithelial ovarian cancer(56). Indeed, high PAI-2 expression in invasive ovarian tumors waslimited to a group of OSPC patients which experienced a more prolongeddisease free and overall survival (56). The reasons for thesedifferences are not clear, but, as previously suggested (57), may berelated at least in part to the actions of macrophage colony stimulatingfactor-1 (CSF-1), a cytokine which has been shown to stimulate therelease of PAI-2 by ovarian cancer cells.

CD24 is a small heavily glycosylated glycosylphosphatidylinositol-linkedcell surface protein, which is expressed in hematological malignanciesas well as in a large variety of solid tumors (58-62). However, it isonly recently that CD24 overexpression has been reported at the RNAlevel in ovarian cancer (39). Consistent with this recent report, in thepresent study CD24 gene was found 14-fold differentially expressed inOSPC when compared to NOVA. Because CD24 is a ligand of P-selectin, anadhesion receptor on activated endothelial cells and platelets, itsexpression may contribute to the metastasizing capacities ofCD24-expressing ovarian tumor cells (63-65). Importantly, CD24expression has recently been reported as an independent prognosticmarker for ovarian cancer patient survival (64). That data combined withthe present findings further suggest that this marker may delineateaggressive ovarian cancer disease and may be targeted for therapeuticand/or diagnostic purposes.

Among the genes identified herein, lipocalin-2 has not been previouslylinked to ovarian cancer. Lipocalin-2 represents a particularlyinteresting marker because of several features. Lipocalins areextracellular carriers of lipophilic molecules such as retinoids,steroids, and fatty acid, all of which may play important roles in theregulation of epithelial cell growth (66,67). In addition, becauselipocalin is a secreted protein, it may play a role in the regulation ofcell proliferation and survival (66,67). Of interest, two recentpublications on gene expression profiling of breast and pancreaticcancer have proposed lipocalin-2 as a novel therapeutic and diagnosticmarker for prevention and treatment of these diseases (66,67). On thebasis of the present findings, lipocalin-2 may be added to the knownmarkers for ovarian cancer.

Osteopontin (SPP1) is an acidic, calcium-binding glycophosphoproteinthat has recently been linked to tumorigenesis in several experimentalanimal models and human patient studies (68,69,70). Because of itsintegrin-binding arginine-glycine-aspartate (RDG) domain and adhesiveproperties, osteopontin has been reported to play a crucial role in themetastatic process of several human tumors (68,71). However, it is onlyrecently that the upregulated expression of osteopontin in ovariancancer has been identified (72). Importantly, because of the secretednature of this protein, osteopontin has been proposed as a novelbiomarker for the early recognition of ovarian cancer (72). In thisstudy, the SPP1 gene was found 10-fold differentially expressed in OSPCwhen compared to NOVA. Taken together these data confirm a highexpression of osteopontin in OSPC.

The organization of kallikreins, a gene family now consisting of 15genes which all encode for trypsin-like or chymotrypsin-like serineproteases, has been recently elucidated (73). Serine proteases have beendescribed to have well characterized roles in diverse cellularactivities, including blood coagulation, wound healing, digestion, andimmune responses, as well as tumor invasion and metastasis (reviewed in73). Importantly, because of the secreted nature of some of theseenzymes, prostate-specific antigen (PSA) and kallikrein 2 have alreadyfound important clinical application as prostate cancer biomarkers (73).Of interest, kallikrein 6 (also known as zyme/protease M/neurosin),kallikrein 10 and matriptase (TADG-15/MT-SP1), were all found to behighly differentially expressed genes in OSPC when compared to NOVA.These data confirm previous results showing high expression of severalkallikrein genes and proteins in ovarian neoplasms (73,74,75,76,77).Moreover, these results obtained by high-throughput technologies forassaying gene expression further emphasize the view that some members ofthe kallikrein family have the potential to become novel cancer markersfor early diagnosis of ovarian cancer (77) as well as targets for noveltherapies against recurrent/refractory ovarian disease (78). Otherhighly ranked genes in OSPC included stratifin, desmoplakin, S100A2,cytokeratins 6 and 7, and MUC-1.

A large number of down-regulated (at least 5-fold) genes in OSPC versusNOVA such as transforming growth factor beta receptor III,platelet-derived growth factor receptor alpha, SEMACAP3, ras homologgene family member I (ARHI), thrombospondin 2 anddisabled-2/differentially expressed in ovarian carcinoma 2 (Dab2/DOC2)have been identified in this analysis. Some of these genes encode forwidely-held tumor suppressor genes such as SEMACAP3, ARHI, and Dab2/DOC2(79), others for proteins important for ovarian tissue homeostasis orthat have been previously implicated in apoptosis, proliferation,adhesion or tissue maintenance.

The identification of TROP-1/Ep-CAM and CPE epithelial receptors as someof the most highly differentially expressed genes in OSPC compared toNOVA indicate that novel therapeutic strategies targeting TROP-1/Ep-CAMby monoclonal antibodies and/or claudin-3 and 4 by local and/or systemicadministration of Clostridium Perfringens enterotoxin can be aneffective therapeutic modalities in patients harboring OSPC refractoryto standard treatment.

Conclusions

Gene expression data identified 129 and 170 genes that exhibited >5-foldup-regulation and down-regulation, respectively, in primary OSPC whencompared to NOVA. Genes overexpressed in established OSPC cell lineswere found to have little correlation with those overexpressed inprimary OSPC, highlighting the divergence of gene expression that occursas the result of long-term in vitro growth. Hierarchical clustering ofthe expression data readily distinguished normal tissue from primaryOSPC. Laminin, claudin-3 and claudin-4, tumor-associated calcium signaltransducer 1 and 2 (TROP-1/Ep-CAM; TROP-2), ladinin 1, S100A2, SERPIN2(PAI-2), CD24, lipocalin 2, osteopontin, kallikrein 6 (protease A) andkallikrein 10, matriptase (TADG-15) and stratifin were found among themost highly overexpressed genes in OSPC when compared to NOVA.Differential expression of some of these genes including claudin-3 andclaudin-4, TROP-1 and CD24 was validated by quantitative RT-PCR as wellas by flow cytometry on primary OSPC and NOVA. Immunohistochemicalstaining of formalin fixed paraffin embedded tumor specimens from whichprimary OSPC cultures were derived further confirmed differentialexpression of CD24 and TROP-1/Ep-CAM markers on OSPC vs. NOVA.

Example 2 Gene Expression Fingerprint of Uterine Serous PapillaryCarcinoma: Identification of Novel Molecular Markers for Uterine SerousCancer Diagnosis and Therapy

The goal of this study was to identify genes with a differential patternof expression between uterine serous papillary carcinoma (USPC) andnormal endometrial cells (NEC) and to use this knowledge to developnovel diagnostic and therapeutic markers for uterine cancer.Oligonucleotide microarrays were used to interrogate the expression of10,000 known genes to analyze gene expression profiling of 10 highlypurified USPC cultures and 5 primary NEC. mRNA fingerprints readilydistinguished USPC from normal endometrial epithelial cells andidentified a number of genes highly differentially expressed between NECand USPC.

Materials and Methods

Establishment of USPC and NEC Primary Cell Lines. A total of fifteenprimary cell lines (i.e., 10 USPC and 5 NEC) were established aftersterile processing of samples from surgical biopsies collected between1997 and 2003 at the University of Arkansas for Medical Sciences, aspreviously described for USPC specimens (80) and normal endometrial cellcultures (81,82). All fresh samples were obtained with appropriateconsent according to IRB guidelines. Tumors were staged according to theF.I.G.O. operative staging system. A total abdominal hysterectomy withbilateral salpingo oophorectomy and bilateral pelvic lymphadenectomy wasperformed in all uterine carcinoma patients while normal endometrialtissue was obtained from consenting donors undergoing surgery for benignpathology. No patient received chemotherapy or radiation before surgery.The patient characteristics are described in Table 3. Briefly, normaltissue was obtained from healthy endometria, mechanically minced andenzymatically dissociated with 0.14% collagenase Type I (Sigma, St.Louis, Mo.) in RPMI 1640 as described previously by Bongso et al. withminor modifications (81). After 1-2 hrs incubation with enzyme on amagnetic stirring apparatus at 37° C. in an atmosphere of 5% CO₂, theresulting suspension was collected by centrifugation at 100 g for 5-10minutes and washed twice with RPMI 1640 medium (Sigma) containing 10%fetal bovine serum (FBS, Invitrogen, Grand Island, N.Y.). The finalpellet was then placed in RPMI 1640 (Sigma) containing 10% FBS, 200 u/mlpenicillin, and 200 μg/ml streptomycin in tissue culture flasks or Petridishes (Invitrogen). The epithelial explants were then allowed to attachand proliferate. Explants were trypsinized and subcultured for 1 to 2passages before being collected for RNA extraction. Tumor tissue wasmechanically minced in RPMI 1640 to portions no larger than 1-3 mm³ andwashed twice with RPMI 1640. The portions of minced tumor were thenplaced into 250 ml flasks containing 30 ml of enzyme solution [0.14%collagenase Type I (Sigma) and 0.01% DNAse (Sigma, 2000 KU/mg)] in RPMI1640, and either incubated on a magnetic stirring apparatus for 1-2 hrsat 37° C. in an atmosphere of 5% CO₂ or overnight at 4° C. Enzymaticallydissociated tumor was then filtered through 150 μm nylon mesh togenerate a single cell suspension. The resultant cell suspension wasthen washed twice in RPMI 1640 plus 10% FBS. Primary cell lines weremaintained in RPMI 1640, supplemented with 10% FBS, 200 u/ml penicillin,200 μg/ml streptomycin at 37° C., 5% CO₂. Tumor cells were collected forRNA extraction at a confluence of 50% to 80% after a minimum of two to amaximum of ten passages in vitro. The epithelial nature and the purityof USPC and NEC cultures was verified by immunohistochemical stainingand flow cytometric analysis with antibodies against cytokeratin andvimentin as previously described (80,82). Only primary cultures whichhad at least 90% viability and contained >99% epithelial cells were usedfor total RNA extraction.

TABLE 3 Patient Age Stage USPC 1 65 IV B USPC 2 75 III C USPC 3 75 IV AUSPC 4 59 IV A USPC 5 59 III C USPC 6 62 IV B USPC 7 63 III C USPC 8 61III C USPC 9 78 III C USPC 10 64 IV A

Gene Cluster/Treeview. The hierarchical clustering of average-linkagemethod with the centered correlation metric was used (35). For theunsupervised hierarchical clustering shown in FIG. 4, a total of 7,328probe sets were scanned across 10 USPCs and 5 NECs. The 7,328 probe setswere derived from 12,588 by filtering out all control genes, all geneswith absent detections, and genes not fulfilling the test of standarddeviation greater than 0.5 (0.5 being the log base 2 of the signal).Table 4 shows only genes significantly expressed by both WRS and SAManalyses and whose average change in expression level was at leastfive-fold.

Results

Gene Expression Profiles Distinguish USPC from NEC and IdentifyDifferentially Expressed Genes. Tumor tissue flash frozen biopsies areknown to contain significant numbers of contaminant stromal cells aswell as a variety of host derived immune cells (e.g., monocytes,dendritic cells, lymphocytes). In addition, USPC represent rare tumorswhich may present in either pure forms, or admixed with endometrioid orclear cell tumor cells (i.e., mixed USPC) (9,10). To minimize the riskof contamination of USPC RNA with that of normal cells or tumor cellswith different histology (i.e., endometrioid or clear cells), as well asto reduce the complexity of gene expression data analysis, in this studyRNA was extracted only from short term primary tumor cell culturescollected only from USPC with single type differentiation. Short termUSPC and NEC cell cultures, minimizing the risk of a selection biasinherent in any long term in vitro growth, may provide an opportunity tostudy differential gene expression between highly enriched populationsof normal and tumor-derived epithelial cells. Accordingly, comprehensivegene expression profiles of 10 primary USPC and 5 primary NEC cell lineswere generated using high-density oligonucleotide arrays with 12,533probe sets, which in total interrogated some 10,000 genes.Characteristics of the 10 patients from whom the primary USPC cell lineswere derived are shown in Table 3. Using unsupervised hierarchicalcluster analysis with 7,238 probe sets, differences in gene expressionbetween USPC and NEC were identified that readily distinguished the twogroups of primary cultures. As shown in FIG. 4, all 10 USPC were foundto group together in the rightmost columns of the dendrogram. Similarly,in the leftmost columns all 5 NEC were found to cluster tightlytogether. After filtering out most “absent” genes, the SAM and thenonparametric WRS test (p<0.05) were performed to identify genesdifferentially expressed between USPC and NEC. A total of 2,829 probesets were found differentially expressed between USPC and NEC withp<0.05 by WRS and with a median FDR of 0.35% and a 90th percentile FDRof 0.59% by SAM. Of the 2,829 aforementioned probe sets, there were 529probe sets showing >5-fold change. As shown in Table 4, a group of 139probe sets were found highly expressed in USPC and underexpressed inNEC. Included in this group of genes are CDKN2A/p16/p14ARF, L1 celladhesion molecule (L1CAM), claudin-3 (CLDN3) and claudin-4 (CLDN4),kallikrein 6 (protease A) and kallikrein 10 (NES1), interleukin-6,interleukin-18 and plasminogen activator receptor (PLAUR) (Table 4).Importantly, c-erbB2 was 14-fold more highly expressed in USPC than inNEC (Table 4). The second profile was represented by 390 genes that werehighly expressed in NEC and underexpressed in USPC (data not shown).Included in this group of genes are transforming growth factor betareceptor III, platelet-derived growth factor receptor alpha, SEMACAP3,ras homolog gene family, member I (ARHI), and differentiallydownregulated in ovarian carcinoma 1 (DOC1).

TABLE 4 Upregulated genes expressed at least 5-fold higher in USPCcompared with NEC Ratio Probe Set Gene Symbol SAM Score(d) p of wrsuspc/nec 1713_s_at CDKN2A 10.59223007 0.0027 101.9077377 36288_at KRTHB14.573430656 0.0027 77.3983986 33272_at SAA1 3.777977393 0.006345.74937337 41294_at KRT7 7.173346265 0.0027 41.46788873 32154_at TFAP2A7.636996321 0.0027 32.47929396 31610_at MAP17 3.621151787 0.009330.28302802 408_at — 4.070053148 0.0063 30.14111158 32821_at LCN25.126089463 0.0027 27.69975608 35174_i_at EEF1A2 2.839620426 0.027826.80482891 38551_at L1CAM 3.115032534 0.0196 25.60938089 38249_at VGLL15.273984976 0.0027 24.69495091 35879_at GAL 5.593811144 0.002723.48953559 36838_at KLK10 3.455062978 0.0136 23.17518549 38299_at IL63.62957424 0.0041 19.05873079 38051_at MAL 4.877642645 0.004117.51555106 41469_at PI3 2.853526521 0.0063 16.90464558 40412_at PTTG15.218191198 0.0027 16.61222352 1886_at WNT7A 3.544426758 0.019616.11519778 33128_s_at CST6 4.221666931 0.0136 15.97856318 38414_atCDC20 7.317470579 0.0027 15.64601435 34012_at KRTHA4 2.410988057 0.027815.37247475 37554_at KLK6 3.784630357 0.0093 15.23781352 1802_s_at ERBB22.566389361 0.0136 14.52012028 41060_at CCNE1 6.092165808 0.002714.16647068 36837_at KIF2C 6.129605781 0.0027 14.1328483 34213_at KIBRA5.300586641 0.0027 13.27228177 1651_at UBE2C 5.554093545 0.002712.87617243 35276_at CLDN4 6.381184288 0.0027 12.74825421 36990_at UCHL14.623383279 0.0027 12.30505908 35977_at DKK1 4.494993915 0.004112.25382636 36113_s_at TNNT1 4.071523595 0.0027 11.93824813 2011_s_atBIK 3.451043397 0.0063 11.66959681 543_g_at CRABP1 3.193471228 0.009311.55494382 34852_g_at STK6 6.224691811 0.0027 11.51812047 33483_at NMU4.093975777 0.0027 11.42057993 39109_at TPX2 6.161639109 0.002711.29208457 37018_at HIST1H1C 2.26997194 0.0278 10.74270622 1165_at IL183.220966429 0.0041 10.65596528 36477_at TNNI3 2.867426116 0.013610.61101382 572_at TTK 3.720282658 0.0093 9.723902052 31542_at FLG2.622102112 0.0196 9.600831601 35937_at MICB 4.238382451 0.00939.460109582 36155_at SPOCK2 2.277735266 0.0278 9.216570003 32186_atSLC7A5 4.148798845 0.0063 9.121679665 35766_at KRT18 5.933225457 0.00279.01220054 35822_at BF 3.266560726 0.0063 8.952514469 35714_at PDXK6.549900892 0.0027 8.898191704 1369_s_at — 2.679010624 0.01968.773380878 40079_at RAI3 4.515766371 0.0063 8.626843209 37168_at LAMP32.837727959 0.0136 8.616807346 39704_s_at HMGA1 5.322233414 0.00278.597894471 1887_g_at WNT7A 3.003097491 0.0196 8.491813649 36929_atLAMB3 5.769944566 0.0027 8.354149098 527_at CENPA 6.125858747 0.00278.32992789 41081_at BUB1 4.882654417 0.0027 8.213759056 885_g_at ITGA34.447267172 0.0027 8.20660555 2021_s_at CCNE1 4.399072926 0.00418.199388463 33904_at CLDN3 3.296023945 0.0136 8.020010794 33730_at RAI34.648262631 0.0041 7.923899439 34736_at CCNB1 5.077963775 0.00637.896644626 757_at ANXA2 3.514460359 0.0063 7.870466864 910_at TK13.933693732 0.0093 7.869091533 34851_at STK6 4.491412407 0.00417.764803777 34703_f_at — 2.275488598 0.0278 7.710260816 34715_at FOXM15.318031066 0.0027 7.659023602 38971_r_at TNIP1 6.799881197 0.00277.595036872 32263_at CCNB2 4.245537907 0.0063 7.578513543 1680_at GRB74.013375211 0.0027 7.471384928 38247_at F2RL1 3.185259514 0.00937.432476326 160025_at TGFA 6.08814344 0.0027 7.355344272 1945_at CCNB15.297806506 0.0041 7.291039832 31792_at ANXA3 4.872657477 0.00417.266892828 182_at ITPR3 5.431705752 0.0027 7.172450367 1117_at CDA2.936875649 0.0093 7.114518646 902_at EPHB2 5.186069433 0.00277.065363569 634_at PRSS8 5.21560703 0.0041 7.001894703 41169_at PLAUR3.982498409 0.0063 7.00139089 33203_s_at FOXD1 3.4642857 0.00936.989749222 40095_at CA2 4.285159359 0.0027 6.946396937 38940_at AD0245.064744169 0.0041 6.928406028 34348_at SPINT2 6.262957935 0.00276.877224695 33933_at WFDC2 3.343526736 0.0136 6.820073691 35281_at LAMC23.346662529 0.0093 6.7580474 349_g_at KIFC1 5.275031682 0.00416.700913018 33218_at ERBB2 2.710053625 0.0027 6.615105998 38881_i_atTRIM16 3.000641338 0.0196 6.506893575 1536_at CDC6 4.666139295 0.00416.463305623 38482_at CLDN7 4.930843791 0.0041 6.409117877 40697_at CCNA23.396480338 0.0093 6.40768505 41688_at TM4SF11 4.390330663 0.00276.366861533 38158_at ESPL1 6.007466409 0.0027 6.225688779 38474_at CBS3.379648389 0.0093 6.212078913 36483_at GALNT3 3.889728637 0.00416.181109111 35372_r_at IL8 2.359705895 0.0278 6.133149591 41585_atKIAA0746 4.436299723 0.0027 6.092207586 36832_at B3GNT3 5.4569676670.0027 5.941291793 1107_s_at G1P2 3.937533177 0.0063 5.92328701935207_at SCNN1A 3.076038486 0.0136 5.920739634 36863_at HMMR 2.8300015860.0196 5.905038013 38631_at TNFAIP2 4.924314508 0.0027 5.89774564236813_at TRIP13 5.665655915 0.0027 5.870351247 41048_at PMAIP13.489974054 0.0062 5.853172336 2084_s_at ETV4 3.742551143 0.00935.798002338 33245_at MAPK13 3.774897977 0.0136 5.766618762 37347_atCKS1B 5.542650247 0.0027 5.762817533 34282_at NFE2L3 2.668167751 0.01365.734907375 330_s_at — 4.026422371 0.0041 5.726752495 41732_at na6.920337146 0.0027 5.706487141 1516_g_at — 6.725730866 0.0027 5.63870137904_s_at TOP2A 3.418887485 0.0063 5.634251452 36041_at EXO1 4.9708409160.0027 5.59235892 33143_s_at SLC16A3 4.007293245 0.0063 5.5659145737228_at PLK 4.500601808 0.0041 5.564532365 1854_at MYBL2 4.1167126520.0063 5.54317592 40407_at KPNA2 4.188947411 0.0041 5.51635645 33282_atLAD1 3.904051584 0.0063 5.509367036 40145_at TOP2A 3.30652637 0.00935.48127065 1100_at IRAKI 5.530078337 0.0027 5.470162749 37883_i_atAF038169 3.159630542 0.0027 5.460495655 37343_at ITPR3 5.2577212510.0027 5.449013729 31598_s_at GALE 4.646763029 0.0027 5.442955253 889_atITGB8 2.743766192 0.0093 5.370592815 37558_at IMP-3 3.122846843 0.00935.364127468 32715_at VAMP8 5.685902454 0.0027 5.352873419 36312_atSERPINB8 3.611288676 0.0027 5.327343554 37210_at INA 3.550708512 0.00635.307526088 35699_at BUB1B 3.664553007 0.0196 5.279075308 32787_at ERBB32.657607539 0.0041 5.247404657 32275_at SLPI 3.726091901 0.00415.221163981 893_at E2-EPF 3.774672918 0.0063 5.196412396 41583_at FEN15.481105111 0.0027 5.196005796 41781_at PPFIA1 4.113488223 0.00275.194931774 40726_at KIF11 2.94101083 0.0093 5.1806793 41400_at TK14.245983179 0.0093 5.167172588 41409_at C1orf38 3.109232321 0.00635.100239097 40425_at EFNA1 2.738432716 0.0196 5.067718102 32081_at CIT6.162032917 0.0027 5.043567722 1108_s_at EPHA1 4.863995126 0.00275.040980858 33338_at STAT1 3.274771895 0.0063 5.029498048

Validation of the Microarray Data. Quantitative-RT-PCR assays were usedto validate the microarray data. Seven highly differentially expressedgenes between USPC and NEC (i.e., CDKN2A/p16, CDKN2A/p14ARF, L1CAM,claudin-3, claudin-4, GRB-7 and c-erbB2) were selected for q-RT-PCRanalysis. A comparison of the microarray and q-RT-PCR data for thesegenes is shown in FIG. 5. Expression differences between USPC and NECfor CDKN2A/p16 (p=0.002), CDKN2A/p14ARF (p=0.002), L1CAM, (p=0.01),claudin-3 (p=0.01), claudin-4 (p=0.002) GRB-7 (p=0.002) and c-erbB2(p=0.01) were readily apparent (Table 4 and FIG. 4). Moreover, for allseven genes tested, the q-RT-PCR data were highly correlated to themicroarray data (p<0.001) (r=0.81, 0.80, 0.75, 0.69, 0.82, 0.71 and0.65, respectively), with all the samples (i.e., 10 USPC and 5 NEC)included in both the q-RT-PCR and microarray experiments. Thus, q-RT-PCRdata suggest that most array probe sets are likely to accurately measurethe levels of the intended transcript within a complex mixture oftranscripts.

Claudin-4 Expression by Immunohistology on USPC and NEC Tissue Blocks.To determine whether the high or low expression of the claudin-4 genedetected by microarray and q-RT-PCR assays in primary USPC and NEC celllines, respectively, is the result of a selection of a subpopulation ofcancer cells present in the original tumor, or whether in vitroexpansion conditions may have modified gene expression,immunohistochemical analysis of claudin-4 protein expression on formalinfixed tumor tissue was performed on the uncultured primary surgicalspecimens from which the USPC cell lines were derived. Both cytoplasmicand membranous staining for claudin-4 protein expression was noted inthe majority of USPC specimens (i.e., 90% score 3+ and 2+) (Table 5,micrographs not shown). In contrast, only low levels of membranousstaining for claudin-4 protein was found in the NEC tissue samplestested by immunohistochemistry (Table 5, p=0.02 USPC vs. NEC by studentt test). To confirm and validate the immunohistochemical results in anindependent series of USPC, formalin fixed tumor tissue blocks from 8further surgical specimens (i.e., USPC 11 to 18, Table 5) similarlyobtained from patients harboring advanced stage disease were tested forclaudin-4 expression. Again, heavy cytoplasmic and/or membranousstaining for the claudin-4 receptor was found in the striking majorityof the further USPC sample tested.

TABLE 5 Claudin-4 staining Claudin-4 Patient positivity NEC 1 1+ NEC 21+ NEC 3 1+ NEC 4 1+ NEC 5 1+ USPC 1 3+ USPC 2 3+ USPC 3 3+ USPC 4 2+USPC 5 3+ USPC 6 2+ USPC 7 3+ USPC 8 1+ USPC 9 3+ USPC 10 3+ USPC 11 3+USPC 12 3+ USPC 13 1+ USPC 14 2+ USPC 15 3+ USPC 16 2+ USPC 17 3+ USPC18 2+

Discussion

Hierarchical clustering of the samples and gene expression levels withinthe samples led to the unambiguous separation of USPC from NEC. Thisstudy identified 529 genes differentially expressed between USPC and NECwhose average change in expression level between the two groups was atleast five-fold and which were found significant with both WRS test andSAM analysis. The known function of some of these genes may provideinsights in the molecular pathogenesis and the highly aggressivebiologic behavior of uterine serous tumors while others may prove to beuseful diagnostic and therapeutic markers against this disease.

For example, the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene wasfound to be the most highly differentially expressed gene in USPC withover 101-fold up-regulation relative to NEC. Importantly, the CDKN2Agene is a putative oncosuppressor gene encoding two unrelated proteins,both cellular growth inhibitors, in different reading frames (83). Oneis p16, which regulates retinoblastoma protein (pRb)-dependent G1arrest, and the second is p14ARF, which blocks MDM2-induced p53degradation, resulting in an increase in p53 levels and consequent cellcycle arrest (83). Although loss of p53 function is considered criticalfor the molecular pathogenesis of USPC (84,85), it is only recently thatabnormality of the Rb pathway has been suggested to define a subgroup ofaggressive endometrial carcinomas with poor prognosis (86). QuantitativeRT-PCR analysis of expression of both p16 and p14ARF in the USPC seriesfound extremely high levels of both transcripts, suggesting that themarked overexpression of the CDKN2A gene may be attributable to anegative feedback loop due to the loss of function of both pRb and p53proteins (84,85,86). Consistent with this view, an inverse relationshipbetween the expression of p16 and p14ARF proteins and the presence ofnormal or functional Rb and p53 in human cancer cells has beenpreviously demonstrated (87,88). Thus, these data suggest for the firsttime that CDKN2A gene overexpression may represent a consistent geneticanomaly of USPC secondary to an autoregulatory feedback loop due todisruption of both the p16-CDK4/cyclin D1-pRb pathway and thep14ARF-MDM2-p53 pathway.

Among the genes identified here, lipocalin-2 has not been previouslylinked to uterine cancer. Lipocalins are extracellular carriers oflipophilic molecules such as retinoids, steroids, and fatty acid, all ofwhich may play important roles in the regulation of epithelial cellgrowth (70,71). In addition, because lipocalin is a secreted protein, itmay play a role in the regulation of cell proliferation and survival(70,71). Of interest, two recent publications on gene expressionprofiling of breast and pancreatic cancer have proposed lipocalin-2 as anovel therapeutic and diagnostic marker for prevention and treatment ofthese diseases (89,90). On the basis of the present findings,lipocalin-2 may be added to the known markers for USPC.

Among the several potential therapeutic target gene products identified,genes encoding tight junction (TJ) proteins claudin-3 and claudin-4 wereconsistently found as two of the most highly up-regulated genes in USPC,with over 8 and 12-fold up-regulation, respectively, relative to NEC.Although the exact function of claudin-3 and claudin-4 in USPC is stillunclear, these proteins have been shown to represent the epithelialreceptors for Clostridium perfringens enterotoxin (CPE), and to be theonly family members of the transmembrane tissue-specific claudinproteins capable of mediating CPE binding and cytolysis. Proteinexpression data obtained by immunohistochemistry with anti-claudin-4antibody on USPC blocks further support the proposal that claudins mayrepresent therapeutic targets.

The organization of kallikreins, a gene family now consisting of 15genes that encode for trypsin-like or chymotrypsin-like serineproteases, has been recently elucidated (77). Serine proteases have wellcharacterized roles in diverse cellular activities, including bloodcoagulation, wound healing, digestion, and immune responses, as well astumor invasion and metastasis (reviewed in 77). Secreted serineproteases such as prostate-specific antigen (PSA) and kallikrein 2 havealready found important clinical application as prostate cancerbiomarkers (77). Of interest, kallikrein 6 (also known as zyme/proteaseM/neurosin), and kallikrein-10 (NES1), two serine proteases recentlyshown to be present at high levels in the circulation of a subset ofovarian cancer patients (80,81,91), were both highly differentiallyexpressed genes in USPC when compared to NEC. Kallikrein 6 andkallikrein 10 overexpression has been shown to correlate with intrinsicresistance to adjuvant chemotherapy and with a poor prognosis in ovariancancer patients (81,014). These data are thus consistent with thepresent results showing high expression of kallikreins 6 and kallikrein10 in USPC, a variant of endometrial carcinoma characterized by anaggressive biologic behavior and an inborn resistance to chemotherapy(7,8,9,10,12).

c-erbB2 gene was found to be one of the most highly differentiallyexpressed genes in USPC with over 14-fold up-regulation compared withNEC. Furthermore, the growth factor receptor-bound protein 7 (GRB7), agene tightly linked to c-erbB2 and previously reported coamplified andcoexpressed with this gene in several cancer types (92) was also highlydifferentially expressed in USPC compared to NEC. These data confirm ourrecent discovery of a striking overexpression of the c-erbB2 geneproduct HER2/neu on 80% of pure USPC (93). Thus, HER2/neu overexpressionmay represent a distinctive molecular marker that in addition to havingthe potential to facilitate differentiation of USPC from thehistologically indistinguishable high grade serious ovarian tumors, mayalso provide insights into the disproportionately poor prognosis of USPCpatients (94,95,96,97). Previous studies have reported that HER2/neuoverexpression in USPC patients may be associated with resistance tochemotherapeutic drugs and shorter survival (96,97). However, highoverexpression of the c-erbB2 gene on USPC provides support for thenotion that trastuzumab (HERCEPTIN™, Genentech, San Francisco, Calif.),a humanized anti-HER-2/Neu antibody that is showing great promise fortreatment of metastatic breast cancer patients overexpressing HER-2/Neuprotein (98), may be a novel, potentially highly effective therapyagainst USPC. Consistent with this view, high sensitivity of USPCnatural killer (NK) cell-mediated antibody-dependent cytotoxicitytriggered by anti-HER-2/Neu-specific antibody in vitro (93), as well asclinical responses in vivo (99) have recently been reported with the useof Herceptin in USPC patients.

L1 adhesion molecule (L1CAM), is a 200-220 kD type I membraneglycoprotein of the immunoglobulin family which plays an importantfunction for development of the nervous system by regulating celladhesion and migration (100). In addition, L1CAM has been recentlyreported to be expressed on a variety of human tumor cell lines such asneuroblastomas, melanomas, and lung carcinomas (101,102,103). Becauseoverexpression of L1CAM by tumor cells may enhance cell migration onvarious extracellular membrane substrates, this molecule has beensuggested to play a role in the adhesion and migration events crucialfor tumor spreading (100,101). In the present analysis, L1CAM was foundto be one of the most highly differentially expressed genes in USPC,with over 25-fold up-regulation relative to NEC. These data are inagreement with a recent report showing high expression of L1CAM in asubset of ovarian and uterine cancers (104). Importantly, patients withL1-positive carcinomas had a poorer prognosis and shorter survival timecompared with patients whose tumors were L1-negative (104), suggesting adirect correlation between L1CAM overexpression and aggressive biologicbehavior. Of interest, however, high levels of soluble L1CAM moleculesresulting from cleavage of its ectodomain by the metalloproteinaseADAM10 (105) have been detected in serum samples of patients withovarian and uterine tumors (104). These observations, combined with theresults herein, support the use of L1CAM as a novel biomarker forprediction of clinical outcome in USPC patients.

Several other highly ranked genes have been identified in this USPC geneexpression profiling analysis including membrane-associated protein 17(MAP17), galanin, urokinase plasminogen activator receptor (UPAR),interleukin-6, forkhead box M1, interleukin-18, dickkopf homolog 1(DKK1), coagulation factor II (thrombin) receptor-like 1, transforminggrowth factor, alpha, interleukin 8, topoisomerase (DNA) II alpha,hyaluronan-mediated motility receptor (RHAMM) and secretory leukocyteprotease inhibitor (antileukoproteinase). For most of the genes founddifferentially expressed in these experiments a correlation with USPCcancer development and progression has not been recognized before. DKK1,for example, has been recently reported by our group to play animportant role in the development of osteolytic lesions in multiplemyeloma (106), but its possible role in USPC pathogenesis and/orprogression has not been elucidated. For other genes such as UPAR, aglycosyl-phosphatidylinositol-anchored glycoprotein whose role inpromoting tumor cell invasion and metastases has been well establishedin a number of experimental studies (107,108,109), a correlation withhigh expression in the uterine serous papillary carcinoma phenotype hasbeen recently reported (110). In this regard, because UPAR proteinexists in two forms, as the glycosyl-phosphatidylinositol-anchoredglycoprotein (50-60 kDa) present on the surface of cells, and as asoluble form of UPAR (sUPAR), produced after cleavage of UPAR byurokinase (35 kDa), measurement of UPAR levels by ELISA, in analogy tobreast cancer (111), can be used as a prognostic marker to identifyearly recurrences in endometrial cancer patients associated with pooroutcome. Finally, the recent demonstration of UPAR as a suitable cancertarget for both therapeutic and diagnostic application by specificantibody directed against its ligand binding domain (112) may provide afoundation for the development of a new type-specific therapy againstthis highly aggressive disease.

A large number of down-regulated (at least 5-fold) genes in USPC versusNEC such as transforming growth factor beta receptor III,platelet-derived growth factor receptor alpha, SEMACAP3, ras homologgene family member I (ARHI), and differentially downregulated in ovariancarcinoma 1 (DOC1) have been identified in this analysis. Some of thesegenes encode for widely-held tumor suppressor genes such as SEMACAP3,ARHI, and DOC1 (113), others for proteins important for tissuehomeostasis or that have been previously implicated in apoptosis,proliferation, adhesion or tissue maintenance.

In conclusion, multiple USPC restricted markers have been identified inthis analysis. Most of these genes have not been previously linked withthis disease and thus represent novel findings. The identification ofHER2/neu and CPE epithelial receptors among the others as some of themost highly differentially expressed genes in USPC when compared to NECindicate that therapeutic strategies targeting HER2/neu by monoclonalantibodies (93,99) and claudin-3 and claudin-4 by local and/or systemicadministration of Clostridium Perfringens enterotoxin can be effectivemodalities for the treatment of patients harboring this highlyaggressive and chemotherapy resistant variant of endometrial cancer.

Conclusions

Unsupervised analysis of mRNA fingerprints readily distinguished USPCfrom NEC and identified 139 and 390 genes that exhibited >5-foldup-regulation and down-regulation, respectively, in primary USPC whencompared to NEC. Many of the genes up-regulated in USPC were found torepresent adhesion molecules, secreted proteins and oncogenes, such asL1 cell adhesion molecule (L1CAM), claudin-3 and claudin-4, kallikrein 6(protease M) and kallikrein 10, (NES1), interleukin-6, interleukin-18,urokinase plasminogen activator receptor (UPAR), and c-erbB2.Down-regulated genes in USPC included transforming growth factor betareceptor III, platelet-derived growth factor receptor alpha, SEMACAP3,ras homolog gene family, member I (ARHI), and differentiallydownregulated in ovarian carcinoma gene 1 (DOC1). Quantitative RT-PCRwas used to validate differences in gene expression between USPC and NECfor some of these genes, including cyclin-dependent kinase inhibitor 2A(CDKN2A/p16 and CDKN2A/p14ARF), L1CAM, claudin-3, claudin-4, GRB-7 andc-erbB2. Finally, expression of the high affinity epithelial receptorfor Clostridium perfringens enterotoxin (CPE) claudin-4, was furthervalidated through immunohistochemical analysis of formalin-fixedparaffin-embedded specimens from which the primary USPC cultures wereobtained, as well as an independent set of archival specimens.

Example 3 Treatment of Chemotherapy-Resistant Human Ovarian CancerXenografts in C.B-17/SCID Mice by Intraperitoneal Administration ofClostridium Perfringens Enterotoxin (CPE)

In this study, real-time PCR was used to quantify the expression levelsof claudin-3 and claudin-4 receptors in several chemotherapy naïve andchemotherapy resistant freshly explanted ovarian tumors. In addition,the ability of CPE to kill chemotherapy sensitive and chemotherapyresistant ovarian cancers overexpressing claudin-3 and/or claudin-4-invitro was tested. Finally, the in vivo efficacy of CPE therapy in SCIDmouse xenografts was studied in a highly relevant clinical model ofchemotherapy resistant freshly explanted human ovarian cancer (i.e.,OVA-1).

Materials and Methods

Cloning and Purification of NH₂ Terminus His-tagged CPE. Clostridiumperfringens strain 2917 obtained from American Type Culture Collection(Manassas, Va.) was grown from a single colony and used to preparebacterial DNA with the INSTAGENE™ kit, according to manufacturer'sdirections (Bio-Rad Lab, Hercules, Calif.). The bacterial DNA fragmentencoding full-length CPE gene (GenBank AJ000766) was PCR amplified(primer 1,5′-AGA TGT TAA TGG ATC CAT GCT TAG TAA CAA m AAA TCC-3′ (SEQID NO:6); primer 2,5′-AAA GCT TTT AAA ATT TTT GAA ATA ATA TTG AAT AAGGG-3′ (SEQ ID NO:7)). The PCR products were digested with therestriction enzymes SphI/HindIII and cloned into a SphI/HindIII-digestedpQE-30 expression vector (Qiagen, Valencia, Calif.) to generate anin-frame NH₂-terminus His-tagged CPE expression plasmid,pQE-30-6×HIS-CPE. His-tagged CPE toxin was prepared frompQE-30-6×HIS-CPE transformed Escherichia coli M15. Transformed bacteriawere grown at 37° C. to 0.3-0.4 optical density at 600 nm, after whichCPE protein expression was induced overnight with 1 mM isopropylB-D-thio-galactoside, and the cells harvested, resuspended in 150 mMNaH₂PO₄, 25 mM Tris-HCL, and 8 M urea pH 8.0 buffer, and lysed bycentrifugation at 10.000 rpm for 30 min. The fusion protein was isolatedfrom the supernatant on a POLY-PREP™ Chromatography column (Bio-Rad).His-tagged CPE was washed with 300 mM NaH₂PO₄, 25 mM Tris-HCl, and 10 Murea pH 6.0, and eluted from the column with 200 mM NaH₂PO₄, 25 mMTris-HCl, and 8 M urea pH 6.0. In order to reduce the level of endotoxinfrom His-tagged CPE protein 10 washings with ice-cold PBS withTRITON-X-114™ (from 1% to 0.1%) and 10 washings with ice-cold PBS alonewere performed. Dialysis (M_(r) 3,500 cutoff dialysis tubing) againstPBS was performed overnight. Purified CPE protein was then sterilized by0.2 μm filtration and frozen in aliquots at −70° C.

Primary and Established Cell Lines. Fresh human ovarian cancer celllines (i.e., eleven chemotherapy naive tumors generated from samplesobtained at the time of primary surgery and six chemotherapy resistanttumors obtained from samples collected at the time of tumor recurrence),and 5 established ovarian cancer cell lines (UCI 101, UCI 107, CaOV3,OVACAR-3, OVARK-5) were evaluated for claudin-3 and claudin-4 expressionby real time-PCR. Patient characteristics from which primary specimenswere obtained are depicted in Table 6. Three of the six ovarian tumorspecimens found resistant to chemotherapy in vivo, including OVA-1, afresh ovarian serous papillary carcinoma (OSPC) used to establishovarian xenografts in SCID mice, were confirmed to be highly resistantto multiple chemotherapeutic agents when measured as percentage cellinhibition (PCI) by in vitro Extreme Drug Resistance (EDR) assay(Oncotech Inc. Irvine, Calif.) (114) (data not shown). UCI-101 andUCI-107, two previously characterized and established human serousovarian cancer cell lines were kindly provided by Dr. Alberto Manetta,University of California, Irvine, while CaOV3, OVACAR-3, were purchasedfrom ATCC (Manassas, Va.), and OVARK-5 was established from a stage 1Vovarian cancer patient, as previously described (115). Other controlcell lines evaluated in the CPE assays included Vero cells, normalovarian epithelium (NOVA), normal endometrial epithelium, normalcervical keratinocytes, primary squamous and adenocarcinoma cervicalcancer cell lines, Epstein-Barr transformed B lymphocytes (LCL), andhuman fibroblasts. With the exception of normal cervical keratinocytesand cervical cancer cell lines which were cultured in serum-freekeratinocyte medium, supplemented with 5 ng/ml epidermal growth factorand 35 to 50 μg/ml bovine pituitary extract (Invitrogen, Grand Island,N.Y.) at 37° C., 5% CO2, all other fresh specimens were cultured in RPMI1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS; GeminiBio-products, Calabasas, Calif.), 200 u/ml penicillin, and 200 μg/mlstreptomycin, as previously described (115,19,116). All samples wereobtained with appropriate consent according to IRB guidelines. Tumorswere staged according to the F.I.G.O. operative staging system. Radicaltumor debulking, including a total abdominal hysterectomy andomentectomy, was performed in all ovarian carcinoma patients whilenormal tissues was obtained from consenting similar age donorsundergoing surgery for benign pathology. Tumors were established aftersterile processing of the samples from surgical biopsies as previouslydescribed for ovarian and cervical carcinoma specimens (115,19,116),while normal ovarian tissue was obtained by scraping epithelial cellsfrom the ovarian surface. Briefly, viable tumor tissue was mechanicallyminced in RPMI 1640 to portions no larger than 1-3 mm³ and washed twicewith RPMI 1640. The portions of minced tumor were then placed into 250ml flasks containing 30 ml of enzyme solution [0.14% collagenase Type Iand 0.01% DNAse 2000 KU/mg; (Sigma)] in RPMI 1640, and incubated on amagnetic stirring apparatus overnight at 4° C. Enzymatically dissociatedtumor was then filtered through 150 μm nylon mesh to generate a singlecell suspension. The resultant cell suspension was then washed twice inRPMI 1640 plus 10% FBS. The epithelial nature and the purity ofepithelial tumor cultures was verified by immunohistochemical stainingand flow cytometric analysis with antibodies against cytokeratin aspreviously described (115,19,116). RNA extraction was performed at atumor cell confluence of 50% to 80% after a minimum of two to a maximumof ten passages in vitro. Only primary cultures which had at least 90%viability and contained >99% epithelial cells were used for sensitivityto CPE in vitro.

TABLE 6 Patient Age Grade Stage Histology OSPC 1* 42 G2/3 IV A OSPC OSPC2 67 G3 III B OSPC OSPC 3 61 G3 III C OSPC OSPC 4* 60 G3 III C OSPC OSPC5 59 G2/3 III C OSPC OSPC 6* 72 G3 IV A OSPC OSPC 7 63 G3 III C CC OSPC8 74 G2/3 III C CC OSPC 9 68 G3 III B CC OSPC 10 77 G2/3 III C CC OVA 1165 G3 III C CC OVA 12R 81 G3 IV A OSPC OVA 13R 62 G3 IV A OSPC OVA 14R58 G3 III C OSPC *Patients from which matched chemotherapy naïve andchemotherapy resistant/recurrent disease were both available. OVA-R:Patients with chemotherapy resistant/recurrent disease. OSPC: ovarianserous papillary carcinoma; CC: clear cell ovarian carcinoma.

CPE Treatment of Cell Lines and Trypan Blue Exclusion Test. Tumorsamples and normal control cells were seeded at a concentration of 1×10⁵cells/well into 6-well culture plates (Costar, Cambridge, Mass.) withthe appropriate medium. Adherent tumor samples, fibroblasts and normalepithelial control cell lines were grown to 80% confluence. Afterwashing and renewal of the medium, CPE was added to final concentrationsranging from 0.03 to 3.3 μg/mL. After incubation for 60 minutes to 24hrs at 37° C., 5% CO₂, floating cells were removed and stored, andattached cells were trypsinized and pooled with the floating cells.After staining with trypan blue, viability was determined by countingthe number of trypan blue-positive cells and the total cell number.

SCID Mouse Tumor Xenografts and CPE Treatment. C.B-17/SCID female mice5-7 weeks old (16-18 g in weight) were obtained from HarlanSprague-Dawley (Indianapolis, Ind.) and housed in a pathogen-freeenvironment at the University of Arkansas for Medical Sciences (UAMS).They were given commercial basal diet and water ad libitum. Theexperimental protocol for the use of these animals for these studies wasapproved by the UAMS Institutional Animal Care and Use Committee.Animals were used to generate ovarian tumor xenografts. The OVA-1 cancercell line was injected intraperitoneally (i.p.) at a dose of 5 to7.5×10⁶ into C.B-17/SCID mice in groups of five. In the first set ofexperiments (i.e., large ovarian tumor burden challenge), four weeksafter i.p. tumor injection, mice were injected i.p. with 5.0, 5.5, 6.5μg of CPE dissolved in 1 ml of sterile saline at 72 hrs intervals. In asecond set of experiments, groups of five mice received 7.5 μg or 8.5 μgof CPE i.p. at 72 hrs intervals 1 week after i.p. OVA-1 tumor injectionat a dose of 5×10⁶ tumor cells. All animals were observed twice dailyand weighed weekly, and survival was monitored. In addition, groups ofmice injected i.p. at a dose of 5 to 7.5×10⁶ OVA-1 tumor cells werekilled at one, two, three and four weeks for necropsy and pathologicanalysis. The remaining animals were killed and examined just beforethey died of intraperitoneal carcinomatosis and malignant ascites.

Statistics. Statistical differences in claudin-3 and claudin-4expression between chemotherapy naïve and chemotherapyrecurrent/resistant ovarian tumors were tested using the student t test.For the OVA-1 animal model, survivals were plotted using Kaplan-Meiermethods and compared using the log-rank test. A p value less than 0.05(p<0.05) was used for statistical significance.

Results

Claudin-3 and Claudin-4 Transcript Levels in Chemotherapy Sensitive andChemotherapy Resistant Ovarian Tumors. Quantitative RT-PCR assays wereused to get highly sensitive measurements of claudin-3 and claudin-4expression in normal tissues and fresh and established human tumors.Both claudin-3 and/or claudin-4 genes were highly expressed in allprimary ovarian cancers studied when compared with normal ovarianepithelial cells as well as other normal cells or other gynecologictumors (FIG. 6). Of interest, established ovarian cancer cell lines(UCI-101, UCI 107, CaOV3, OVACAR-3, OVARK-5), were found to express muchlower levels of claudin-3 and/or claudin-4 compared to primary ovariantumors (FIG. 6). Finally, claudin-3 and/or claudin-4 expression wasextremely low in all control tissues examined including normal ovarianepithelium, normal endometrial epithelium, normal cervicalkeratinocytes, and normal human fibroblasts (FIG. 6).

When OSPC collected at the time of primary debulking surgery (6 cases)were compared for claudin-3 and/or claudin-4 receptor expression tothose collected at the time of tumor recurrence after multiple coursesof chemotherapy (6 cases), chemotherapy resistant tumors were found toexpress significantly higher levels of claudin-3 and/or claudin-4receptors (p<0.05, FIG. 7). Importantly, when 3 primary ovarian cancersnaïve to chemotherapy were compared to recurrent ovarian cancersrecovered from the same patients following chemotherapy (i.e., matchedautologous tumor samples), chemotherapy resistant tumors were againfound to express higher levels of claudin-3 and claudin-4 (FIG. 7).

Claudin-3 and Claudin-4 Transcript Levels in Metastatic and PrimaryTumors. Claudin-3 and claudin-4 mRNA copy numbers were quantified in anOVA-1 primary tumor and in a metastatic ovarian tumor (omentummetastasis) later taken from the same patient. The results are shown inFIG. 8. Transcript levels for both claudin-3 and claudin-4 were morethan twice as high in the metastatic cells as in the primary tumor.

Claudin-4 Expression by Immunohistology on OSPC and NOVA Tissue Blocks.To determine whether the high expression of the claudin-4 gene detectedby q-RT-PCR assays in primary ovarian cancer cell lines is the result ofa selection of a subpopulation of cancer cells present in the originaltumor, or whether in vitro expansion conditions may have modified geneexpression, immunohistochemical analysis of claudin-4 protein expressionwas performed on formalin fixed tumor tissue from the uncultured primarysurgical specimens from which fresh ovarian cancers were derived. Asshown in Table 7, moderate to heavy membranous staining for claudin-4protein expression was noted in all the cancer specimens thatoverexpressed the claudin-4 transcript. In contrast, negative or lowstaining was found in all the normal ovarian epithelium tested byimmunohistochemistry (Table 7).

TABLE 7 Claudin-4 staining Claudin-4 Patient positivity NOVA 1 1+ NOVA 21+ OVA 1 3+ OVA 2 3+ OVA 3 3+ OVA 4 2+ OVA 5 3+ OVA 6 2+ OVA 7 3+ OVA 82+ OVA 9 3+ OVA 10 3+ OVA 11 3+

Effects of CPE on Fresh Ovarian and Cervical Cancer Cell Lines andNormal Control Cells. On the basis of the high expression of claudin-3and/or claudin-4 on primary ovarian cancer cell lines, it was suspectedthat ovarian tumors expressing either claudin-3 or claudin-4 would besensitive to CPE-mediated lysis. It was important to demonstrate thisdirectly on fresh human ovarian carcinoma cells, particularly in aclinically relevant setting of ovarian cancer disease for which currentsalvage therapies are ineffective (i.e., chemotherapy resistantdisease). For this reason this study examined short term in vitroprimary cultures of ovarian carcinomas obtained either from chemotherapynaïve patients (i.e., OVA-2, OVA-3 and OVA-5) or patients heavilytreated with different combinations of chemotherapy (i.e., OVA-1, OVA-4and OVA-6) and now with disease progression after multiple chemotherapyregimens. The sensitivity of these primary ovarian tumor cultures toCPE-mediated cytolysis was tested along with an appropriate claudin-3and claudin-4-expressing positive control (i.e., Vero cells),established OSPC cell lines (OVARK-5, CaOV3 and OVACAR-3), and negativecontrols which do not express detectable levels of either claudin-3 orclaudin-4. As shown in FIG. 9, regardless to their sensitivity orresistance to chemotherapy all ovarian tumors tested were foundsensitive to CPE-mediated cytolysis. The cytotoxic effect was dosedependent and was positively correlated to the levels of eitherclaudin-3 or claudin-4 expression as tested by RT-PCR in tumor samples.Importantly, although ovarian tumors demonstrated differentsensitivities to CPE exposure, no ovarian cancer was found viable after24 hrs exposure to CPE at the concentration of 3.3 μg/mL. In contrast,all normal controls tested including ovarian epithelium, cervicalkeratinocytes and mononuclear cells as well as cervical cancer celllines lacking claudin-3 or claudin-4 were not affected by CPE (FIG. 9).

Effect of CPE on Chemotherapy Resistant Ovarian Cancer Cells In Vivo

For in vivo confirmation of the in vitro data, xenograft tumors in SCIDmice were developed by i.p. injection with OVA-1, a primary ovariantumor resistant to multiple chemotherapeutic agents in vitro (by EDRassay) as well as in vivo. Primary OVA-1 tumor cells grew progressivelyas numerous serosal nodules adherent to virtually all intraabdominalorgans (peritoneum, omentum, diaphragm, bowel, liver, pancreas, spleen)and exhibited the capacity for local tissue invasion and formation ofmalignant ascites after 2 to 3 weeks from injection. Tumors firstappeared grossly by the second week as small nodules on the omentum andcontinuously grew to form a confluent omental mass by the time theanimals died (i.e., mean survival 38 days after i.p. injection with7.5×10⁶ OVA-1 cells). Necropsies revealed massive hemorrhagic ascitesand numerous tumor nodules, measuring 1 to 8 mm in diameter, studdingthe entire peritoneal surface and implanting the serosa of virtually allintraabdominal organs.

Previous toxicology studies in mice have reported 0.5 μg/g of CPEadministered i.p. to be a well tolerated and safe dose in 100% of theanimals (i.e., 16.5±1.0 g male SW mice) (117,118). In contrast, someanimals injected with 0.75 μg/g died after CPE injection and all animalsinjected with 1 μg/g of CPE died within 1 to 2 hrs (117,118). Maximumtolerated dose in healthy female mice was determined and is consistentwith these observations (data not shown). In one experiment, groups ofmice harboring large ovarian tumor burden xenografts (i.e., four weeksafter OVA-1 tumor injection) were treated with repeated i.p. CPEinjections every 72 hrs at three different doses (5.0 μg, 5.5 μg or 6.5μg). Control mice harboring OVA-1 received saline alone. CPE injectionswere well tolerated, and no adverse events were observed throughout thecomplete treatment protocol either in control mice receiving CPE aloneor CPE-treated mice harboring large tumor burden. Mice harboring OVA-1treated with saline all died within 6 weeks from tumor injection with amean survival of 38 days (FIG. 10A). In contrast, animals treated withmultiple CPE injections survived significantly longer than controlanimals did (p<0.0001, FIG. 10A). The increase in survival in thedifferent groups of mice treated with the diverse doses of CPE wasclearly dose dependent, with the highest dose injected (i.e., 6.5 μgevery 72 hrs) found to provide the longer survival (FIG. 10A). Inanother set of experiments, mice harboring OVA-1 (a week after tumorinjection with 5×10⁶ cells) were treated with i.p. CPE injections at adose ranging from 7.5 μg to 8.5 μg every 72 hrs. While mice harboringOVA-1 treated with saline all died within 9 weeks from tumor injection(FIG. 10B), three out of five (60%) of mice receiving 7.5 μg doses ofCPE and five out of five (100%) of mice receiving 8.5 μg doses of CPE bymultiple i.p. injections remained alive and free of detectable tumor forthe duration of the study period (i.e., over 120 days, p<0.0001).

Discussion:

In this study, after having confirmed at both the RNA and protein levelsthe expression of high levels of CPE receptors in multiple primaryovarian cancers and tested the in vitro sensitivity of tumor cells toCPE therapy, the efficacy and toxicity of i.p. injection of CPE in vivoin a clinically relevant animal model of chemotherapy resistant ovariantumor xenografts were evaluated.

100% (seventeen out of seventeen) of the primary ovarian cancer celllines tested for claudin-3 and claudin-4 expression by quantitativeRT-PCR overexpress either the high affinity CPE receptor (claudin-4) orthe low affinity CPE receptor (claudin-3). Of interest, all theestablished ovarian cancer cell lines tested (UCI 101, UCI 107, CaOV3,OVACAR-3, OVARK-5), were found to express much lower levels of claudin-3and/or claudin-4 compared to primary ovarian tumors. These data suggestthat prolonged in vitro culture may significantly alter claudin-3 andclaudin-4 gene expression in ovarian cancer. In addition, q-RT-PCRshowed a consistent downregulation of claudin-3 and claudin-4 expressionlevels in the more advanced in vitro passages of primary OSPC (data notshown). Thus, established ovarian cancer cell lines may representsuboptimal models to evaluate the potential of CPE-mediated therapyagainst ovarian cancer in vitro as well as in vivo. Importantly, allprimary ovarian tumors evaluated, including those found to be resistantto chemotherapy in vivo as well as in vitro, were found highly sensitiveto CPE-mediated killing in vitro. In this regard, although ovariantumors demonstrated different sensitivity to CPE exposure, no ovariancancer was found viable after 24 hrs exposure to CPE at theconcentration of 3.3 μg/mL, a dose well tolerated by in vivo i.p.administration of CPE in these experiments. This was in strong contrastwith the lack of sensitivity of normal ovarian epithelium as well asother normal control cells to CPE-mediated cytolysis. These findings arelikely explained by a limited expression of claudin-3 and claudin-4 innormal epithelia compared to ovarian tumor cells.

When the efficacy of multiple i.p. injection of sublethal doses of CPEin vivo in a clinically relevant animal model of chemotherapy resistantovarian tumor xenografts was tested, doses of CPE ranging from 5 to 8.5μg were well tolerated, and no adverse events were observed throughoutthe complete treatment protocol either in control mice receiving CPEalone or CPE-treated mice harboring small and large tumor burden. Thesedata demonstrate that CPE doses found effective in vitro to kill ovariantumor cells may be safely administered i.p. in mice harboring ovariancancer disease. More importantly, survival of mice harboring a largeburden of chemotherapy resistant ovarian disease was significantlyprolonged in a dose dependent manner by repeated i.p. injections of CPE.Finally, when animals harboring 1 week OVA-1 xenografts were treatedwith repeated i.p. injections of CPE, most of the mice remained aliveand free of detectable tumor for the duration of the study (i.e., over120 days). Collectively, these results provide strong evidence thatCPE-based therapy can be useful in the treatment of ovarian cancerpatients refractory to standard treatment modalities.

CPE probably must distribute through a tumor by passive diffusion. Sointraperitoneal CPE administration would likely be most effective inpatients with microscopic residual disease or small tumor burden.

CPE-mediated cytolysis requires only the single step of CPE binding toits receptors and takes place after only a few minutes of tumor cellexposure to toxic CPE concentrations. Thus, the simplicity and rapidityof CPE-mediated cytolysis may result in increased efficacy, reducedopportunity for the development of resistance, and the possibility thathigh local concentrations of CPE may need to be maintained for only arelatively short period of time. Furthermore, because the efficacy ofCPE therapy against chemotherapy resistant ovarian cancer xenografts hasbeen demonstrated in SCID mice (i.e., severely immunocompromisedanimals), it seems unlikely that the host immune system is required toplay a significant role in the in vivo efficacy of CPE therapy. Thispoint is noteworthy because several biologic response modifierspreviously used for the therapy of chemotherapy resistant ovariancancer, including cytokines (119) and humanized monoclonal antibodies(120), unlike CPE therapy, rely for most of their efficacy on theactivation of an uncompromised host immune system (121), a majorlimitation when dealing with elderly ovarian cancer patients heavilypretreated with multiple regimens of immunosuppressive chemotherapy.

In conclusion, it is shown that primary and metastatic ovarian cancersthat have acquired in vivo resistance to chemotherapeutic drugs aresusceptible to killing by CPE-mediated therapy in vitro as well as invivo.

Conclusions

100% (17 out of 17) of the primary ovarian tumors tested overexpressedone or both CPE-receptors by quantitative RT-PCR. All ovarian tumorsdemonstrated a dose-dependent cytotoxic effect to CPE in vitro.Importantly, chemotherapy resistant/recurrent ovarian tumors were foundto express claudin-3 and claudin-4 genes at significantly higher levelswhen compared to chemotherapy naïve ovarian cancers and all primaryovarian tumors tested, regardless of their resistance tochemotherapeutic agents, died within 24 hrs to the exposure to 3.3 μg/mLof CPE in vitro. Metastatic OVA-1 tumors were also found to express moreclaudin-3 and claudin-4 than primary OVA-1 tumors. In addition, the invivo efficacy of intraperitoneal (i.p.) CPE therapy in SCID mousexenografts was studied in a highly relevant clinical model ofchemotherapy resistant freshly explanted human ovarian cancer (i.e.,OVA-1). Repeated i.p. administration of sublethal doses of CPE every 3days significantly inhibited tumor growth in 100% of mice harboring1-week established OVA-1. Repeated i.p. doses of CPE also had asignificant inhibitory effect on tumor progression with extendedsurvival of animals harboring large ovarian tumor burdens (i.e., 4-weeksestablished OVA-1).

Example 4 Production of a Chimeric Antibody Containing a Claudin-BindingPeptide of CPE

In this Example, production of a chimeric antibody having residues290-319 of SEQ ID NO:1 (CPE), which is a peptide of CPE that binds toclaudin-3 and claudin-4, conjugated to the Fc portion of IgG1.

A nucleic acid segment encoding residues 290-319 of SEQ ID NO:1 iscloned into vector pFUSEhFc2(IL2ss) (SEQ ID NO:2) (INVIVOGEN company,www.invivogen.com).

Two single-stranded oligonucleotides 30-CPE-forward (SEQ ID NO:3) and 30CPE-reverse (SEQ ID NO:4) are synthesized. Annealing the two strandsproduces a double-stranded DNA segment with a blunt end and anNcoI-compatible sticky end (a CATG-5′ overhang).

pFUSEhFc2 is digested with EroRV and NcoI. The hybridized insert isligated into the double-digested pFUSEhFc2. The result is a vector thatexpresses a protein with SEQ ID NO:5. Residues 1-21 of SEQ ID NO:5 are asignal peptide of interleukin-2 (ssIL2) for secretion of the expressedprotein in mammalian cells. The signal peptide is cleaved duringsecretion. Residues 23-52 of SEQ ID NO:5 are a claudin-3- andclaudin-4-binding peptide from CPE (residues 290-319 of SEQ ID NO:1).Residues 22 and 53 result from insertion of the CPE residues. Residues54-280 of SEQ ID NO:5 are human Fc (CH2 and CH3 domains and the hingeregion of human IgG1 heavy chain).

The plasmid encoding the 30aaCPE-Fc fusion protein is amplified in E.coli, and then transfected into the murine myeloma cell line Sp2/0 Ag14from the American Type Culture Collection for expression and secretionof the 30aaCPE-Fc fusion protein. The secreted fusion protein ispurified by protein A or protein G affinity chromatography

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All patents, patent documents, and other references cited are herebyincorporated by reference.

1-24. (canceled)
 25. A method of treating cancer in a mammal comprising:administering to the mammal a therapeutically effective amount of acytotoxic agent that binds specifically to claudin-3 and/or claudin-4;wherein the cancer is ovarian cancer or uterine cancer.
 26. The methodof claim 25 wherein the cytotoxic agent is an antibody that bindsspecifically to claudin-3 and/or claudin-4.
 27. The method of claim 25wherein the antibody comprises residues 290-319 of SEQ ID NO:1 or afragment thereof that binds specifically to claudin-3 and/or claudin-4.28. The method of claim 25 wherein the cytotoxic agent comprises (i)residues 290-319 of SEQ ID NO:1 or a fragment thereof that bindsspecifically to claudin-3 and/or claudin-4; coupled to (ii) a cytotoxicmoiety.
 29. An anticancer agent comprising: a moiety that bindsspecifically to claudin-3 and/or claudin-4; coupled to a cytotoxicmoiety; wherein the agent is effective to kill tumor cellsoverexpressing claudin-3 and/or claudin-4 in vitro or in vivo, andwherein the agent is not Clostridium perfringens enterotoxin.
 30. Theanticancer agent of claim 29 wherein the agent is effective to treatcancer in a mammal in vivo, wherein the cancer comprises tumor cellsoverexpressing claudin-3 and/or claudin-4.
 31. The anticancer agent ofclaim 29 wherein the cytotoxic moiety is an anticancer chemotherapeuticagent.
 32. The anticancer agent of claim 29 wherein the cytotoxic moietyis a moiety that stimulates cell killing by immune system components.33. The anticancer agent of claim 32 wherein the cytotoxic moiety is anantibody Fc.
 34. The anticancer agent of claim 32 wherein the cytotoxicmoiety is interleukin-2.
 35. The anticancer agent of claim 29 whereinthe cytotoxic moiety is a therapeutic radionuclide.
 36. The anticanceragent of claim 29 wherein the moiety that binds specifically toclaudin-3 and/or claudin-4 comprises residues 290-319 of SEQ ID NO:1 ora fragment thereof that binds specifically to claudin-3 and/orclaudin-4.